Alpha-amylase variant with altered properties

ABSTRACT

The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits altered properties relative to the parent alpha-amylase.

FIELD OF THE INVENTION

[0001] The present invention relates to a variant of parentTermamyl-like alpha-amylase, which variant has alpha-amylase activityand exhibits an alteration in at least one of the following propertiesrelative to said parent alpha-amylase:

[0002] Substrate specificity, substrate binding, substrate cleavagepattern, thermal stability, pH activity profile, pH stability profile,stability towards oxidation, Ca²⁺ dependency, reduced and increased pIand improved wash performance, specific activity, stability under, e.g.,high temperature and/or low pH conditions, in particular at low calciumconcentrations. The variant of the invention are suitable for starchconversion, ethanol production, laundry wash, dish wash, hard surfacecleaning, textile desizing, and/or sweetner production.

BACKGROUND OF THE INVENTION

[0003] Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis ofstarch and other linear and branched 1,4-glucosidic oligo- andpolysaccharides.

BRIEF DISCLOSURE OF THE INVENTION

[0004] The object of the present invention is to provide a Termamyl-likealpha-amylase, which variant in comparison to the corresponding parentalpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylaseactivity and exhibits an alteration in at least one of the abovementioned properties relative to said parent alpha-amylase.

[0005] Nomenclature

[0006] In the present description and claims, the conventionalone-letter and three-letter codes for amino acid residues are used. Forease of reference, alpha-amylase variants of the invention are describedby use of the following nomenclature:

[0007] Original amino acid(s): position(s): substituted amino acid(s)

[0008] According to this nomenclature, for instance the substitution ofalanine for asparagine in position 30 is shown as:

[0009] Ala30Asn or A30N

[0010] a deletion of alanine in the same position is shown as:

[0011] Ala30* or A30*

[0012] and insertion of an additional amino acid residue, such aslysine, is shown as:

[0013] Ala30AlaLys or A30AK

[0014] A deletion of a consecutive stretch of amino acid residues, suchas amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

[0015] Where a specific alpha-amylase contains a “deletion” incomparison with other alpha-amylases and an insertion is made in such aposition this is indicated as:

[0016] *36Asp or *36D

[0017] for insertion of an aspartic acid in position 36.

[0018] Multiple mutations are separated by plus signs, i.e.:

[0019] Ala30Asp+Glu34Ser or A30N+E34S

[0020] representing mutations in positions 30 and 34 substitutingalanine and glutamic acid for asparagine and serine, respectively.

[0021] When one or more alternative amino acid residues may be insertedin a given position it is indicated as

[0022] A30N,E or

[0023] A30N or A30E

[0024] Furthermore, when a position suitable for modification isidentified herein without any specific modification being suggested, itis to be understood that any amino acid residue may be substituted forthe amino acid residue present in the position. Thus, for instance, whena modification of an alanine in position 30 is mentioned, but notspecified, it is to be understood that the alanine may be deleted orsubstituted for any other amino acid, i.e., any one of:

[0025] R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0026] Further, “A30X” means any one of the following substitutions:

[0027] A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K,A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short:A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

[0028] If the parent enzyme—used for the numbering—already has the aminoacid residue in question suggested for substitution in that position thefollowing nomenclature is used:

[0029] “X30N” or “X30N,V” in the case where for instance one or N or Vis present in the wildtype.

[0030] Thus, it means that other corresponding parent enzymes aresubstituted to an “Asn” or “Val” in position 30.

[0031] Characteristics of Amino Acid Residues

[0032] Charged amino acids:

[0033] Asp, Glu, Arg, Lys, His

[0034] Negatively charged amino acids (with the most negative residuefirst):

[0035] Asp, Glu

[0036] Positively charged amino acids (with the most positive residuefirst):

[0037] Arg, Lys, His

[0038] Neutral amino acids:

[0039] Gly, Ala, Val, Leu, lie, Phe, Tyr, Trp, Met, Cys, Asn, Gln, Ser,Thr, Pro

[0040] Hydrophobic amino acid residues (with the most hydrophobicresidue listed last):

[0041] Gly, Ala, Val, Pro, Met, Leu, lie, Tyr, Phe, Trp,

[0042] Hydrophilic amino acids (with the most hydrophilic residue listedlast):

[0043] Thr, Ser, Cys, Gln, Asn

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is an alignment of the amino acid sequences of five parentTermamyl-like alpha-amylases. The numbers on the extreme left designatethe respective amino acid sequences as follows:

[0045] 1: SEQ ID NO: 4 (SP722)

[0046] 2: SEQ ID NO: 2 (SP690)

[0047] 3: SEQ ID NO: 10 (BAN)

[0048] 4: SEQ ID NO: 8 (BLA)

[0049] 5: SEQ ID NO: 6 (BSG).

DETAILED DISCLOSURE OF THE INVENTION

[0050] The object of the present invention is to provide a Termamyl-likeamylase, which variant has alpha-amylase activity and exhibits analteration in at least one of the following properties relative to theparent alpha-amylase: substrate specificity, substrate binding,substrate cleavage pattern, thermal stability, pH activity profile, pHstability profile, stability towards oxidation, Ca²⁺ dependency,specific activity, stability under, e.g., high temperature and/or low pHconditions, in particular at low calcium concentrations.

[0051] Termamyl-Like Alpha-Amylases

[0052] A number of alpha-amylases produced by Bacillus spp. are highlyhomologous (identical) on the amino acid level.

[0053] The identity of a number of known Bacillus alpha-amylases can befound in the below Table 1: TABLE 1 Percent identity 707 AP1378 BAN BSGSP690 SP722 AA560 Termamyl 707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.0 69.4 BAN 66.9 67.1 100.0 65.667.1 68.8 66.9 80.7 BSG 66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4 SP69087.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2 SP722 86.2 86.6 68.8 67.1 87.2100.0 86.8 70.8 AA560 95.5 86.0 66.9 66.3 87.0 86.8 100.0 68.3 Termamyl68.1 69.4 80.7 65.4 69.2 70.8 68.3 100.0

[0054] For instance, the B. licheniformis alpha-amylase (BLA) comprisingthe amino acid sequence shown in SEQ ID NO: 8 (commercially available asTermamyl™) has been found to be about 81% homologous with the B.amyloliquefaciens alpha-amylase comprising the amino acid sequence shownin SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilusalpha-amylase (BSG) comprising the amino acid sequence shown in SEQ IDNO: 6. Further homologous alpha-amylases include SP690 and SP722disclosed in WO 95/26397 and further depicted in SEQ ID NO: 2 and SEQ IDNO: 4, respectively, herein. Other amylases are the M560 alpha-amylasederived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 anddescribed by Tsukamoto et al., Biochemical and Biophysical ResearchCommunications, 151 (1988), pp. 25-31.

[0055] The KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (fromKAO Corporation).

[0056] Still further homologous alpha-amylases include the alpha-amylaseproduced by the B. lichenformis strain described in EP 0252666. (ATCC27811), and the alpha-amylases identified in WO 91/00353 and WO94/18314. Other commercial Termamyl-like alpha-amylases are comprised inthe products sold under the following tradenames: Optitherm™ andTakatherm™ (Solvay); Maxamyl™ (available from Gist-brocades/Genencor),Spezym AA™ and Spezyme Delta AA™ (available from Genencor), andKeistase™ (available from Daiwa), Dex Io, GC 521 (available fromGenencor) and Ultraphlow (from Enzyme Biosystems).

[0057] Because of the substantial homology found between thesealpha-amylases, they are considered to belong to the same class ofalpha-amylases, namely the class of “Termamyl-like alpha-amylases”.

[0058] Accordingly, in the present context, the term “Termamyl-like”alpha-amylase” is intended to indicate an alpha-amylase, in particularBacillus alpha-amylase, especially Bacillus licheniformis alpha-amylase,which, at the amino acid level, exhibits a substantial identity toTermamyl™, i.e., the B. licheniformis alpha-amylase having the aminoacid sequence shown in SEQ ID NO: 8, herein.

[0059] In other words, all the following alpha-amylases, which has theamino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 13herein are considered to be “Termamyl-like alpha-amylase”. OtherTermamyl-like alpha-amylases are alpha-amylases i) which displays atleast 60%, such as at least 70%, e.g., at least 75%, or at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% homology (identity) with at least one of saidamino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13,and/or is encoded by a DNA sequence which hybridizes to the DNAsequences encoding the above-specified alpha-amylases which are apparentfrom SEQ ID NOS: 1, 3, 5, 7, 9, and of the present specification (whichencoding sequences encode the amino acid sequences shown in SEQ ID NOS:2, 4, 6, 8, 10 and 12 herein, respectively).

[0060] Homology (Identity)

[0061] The homology may be determined as the degree of identity betweenthe two sequences indicating a derivation of the first sequence from thesecond. The homology nay suitably be determined by means of computerprograms known in the art such as GAP provided in the GCG programpackage (described above). Thus, Gap GCGv8 may be used with the defaultscoring matrix for identity and the following default parameters: GAPcreation penalty of 5.0 and GAP extension penalty of 0.3, respectivelyfor nucleic acidic sequence comparison, and GAP creation penalty of 3.0and GAP extension penalty of 0.1, respectively, for protein sequencecomparison. GAP uses the method of Needleman and Wunsch, (1970), J.Mol.Biol. 48,- p.443-453, to make alignments and to calculate the identity.

[0062] A structural alignment between Termamyl (SEQ ID NO: 8) and, e.g.,another alpha-amylase may be used to identify equiva-lent/correspondingpositions in other Termamyl-like alpha-amylases. One method of obtainingsaid structural alignment is to use the Pile Up programme from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading(Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

[0063] Hybridisation

[0064] The oligonucleotide probe used in the characterisation of theTermamyl-like alpha-amylase above may suitably be prepared on the basisof the full or partial nucleotide or amino acid sequence of thealpha-amylase in question.

[0065] Suitable conditions for testing hybridisation involve pre-soakingin 5×SSC and prehybridizing for 1 hour at 40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridisation inthe same solution supplemented with 100 mM ATP for 18 hours at 40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at 75° C. (very high stringency). More details about thehybridisation method can be found in Sambrook et al., Molecular_Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

[0066] In the present context, “derived from” is intended not only toindicate an alpha-amylase produced or producible by a strain of theorganism in question, but also an alpha-amylase encoded by a DNAsequence isolated from such strain and produced in a host organismtransformed with said DNA sequence. Finally, the term is intended toindicate an alpha-amylase, which is encoded by a DNA sequence ofsynthetic and/or cDNA origin and which has the identifyingcharacteristics of the alpha-amylase in question. The term is alsointended to indicate that the parent alpha-amylase may be a variant of anaturally occurring alpha-amylase, i.e., a variant, which is the resultof a modification (insertion, substitution, deletion) of one or moreamino acid residues of the naturally occurring alpha-amylase.

[0067] Parent Termamyl-Like Alpha-Amylases

[0068] According to the invention any Termamy-like alpha-amylase, asdefined above, may be used as the parent (i.e., backbone) alpha-amylase.In a preferred embodiment of the invention the parent alpha-amylase isderived from B. licheniformis, e.g., one of those referred to above,such as the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8.

[0069] Parent Hybrid Termamyl-Like Alpha-Amylases

[0070] The parent alpha-amylase (i.e., backbone alpha-amylase) may alsobe a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises acombination of partial amino acid sequences derived from at least twoalpha-amylases.

[0071] The parent hybrid alpha-amylase may be one, which on the basis ofamino acid homology (identity) and/or DNA hybridization (as definedabove) can be determined to belong to the Termamyl-like alpha-amylasefamily. In this case, the hybrid alpha-amylase is typically composed ofat least one part of a Termamyl-like alpha-amylase and part(s) of one ormore other alpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

[0072] Thus, the parent hybrid alpha-amylase may comprise a combinationof partial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase from which a partial amino acid sequence derives, may beany of the specific Termamyl-like alpha-amylase referred to herein.

[0073] For instance, the parent alpha-amylase may comprise a C-terminalpart of an alpha-amylase derived from a strain of B. licheniformis, anda N-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus (BSG). Forinstance, the parent alpha-amylase may comprise at least 430 amino addresidues of the C-terminal part of the B. licheniformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 10 and an amino acidsegment corresponding to the 445 C-terminal amino add residues of the B.licheniformis alpha-amylase having the amino acid sequence shown in SEQID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical to theTermamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shownin SEQ ID NO: 8, except that the N-terminal 35 amino acid residues (ofthe mature-protein) has been replaced by the N-terminal 33 residues ofBAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylaseshown in SEQ ID NO: 10; or b) an amino acid segment corresponding to the68 N-terminal amino acid residues of the B. stearothermophilusalpha-amylase having the amino add sequence shown in SEQ ID NO: 6 and anamino acid segment corresponding to the 415 C-terminal amino addresidues of the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8.

[0074] Another suitable parent hybrid alpha-amylase is the onepreviously described in WO 96/23874 (from Novo Nordisk) constituting theN-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids1-300 of the mature protein) and the C-terminus from Termamyl (aminoacids 301-483 of the mature protein).

[0075] In a preferred embodiment of the invention the parentTermamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8and SEQ ID NO: 10. Specifically, the parent hybrid Termamyl-likealpha-amylase may be a hybrid alpha-amylase comprising the 445C-terminal amino acid residues of the B. licheniformis alpha-amylaseshown in SEQ ID NO: 8 and the 37 N-terminal amino acid residues of thealpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 10,which may suitably further have the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 8). Thelatter mentioned hybrid is used in the examples below and is referred toas LE174.

[0076] Other specifically contemplated parent alpha-amylase includeLE174 with fewer mutations, i.e., the right above mentioned hydridhaving the following mutations: A181T+N90F+A209V+Q264S;N190F+A209V+Q264S; A209V+Q264S; Q264S; H156Y+N 190F+A209V+Q264S;H156Y+A209V+Q264S; H 156Y+Q264S; H156Y+A181T+A209V+Q264S;H156Y+A181T+Q264S; H156Y+Q264S; H156Y+A181T+N190F+Q264S;H156Y+A181T+N190F; H156Y+A181T+N190F+A209V. These hybrids are alsoconsidered to be part of the invention.

[0077] In a preferred embodiment the parent Termamyl-like alpha amylaseis LE174, SP722, or M560 including any of LE174+G48A+T49I+G107A+I201F;LE174+M197L; LE174+G48A+T49I+G107A+M197L+I201F, or SP722+D183*+G184*;SP722+D183*+G184*+N195F; SP722+D183*+G184*+M202L;SP722+D183*+G184*+N195F+M202L; BSG+I181*+G182*; BSG+I181*+G182*+N193F;BSG+I181*+G182*+M200L; BSG+I181*+G182*+N193F+M200L; AA560+D183*+G184*;AA560+D183*+G184*+N195F; M560+D183*+G184*+M202L;AA560+D183*+G184*+N195F+M202L.

[0078] “BSG+I181*+G182*+N193F” means the B. stearothermophilusalpha-amylase has been mutated as follows: deletions in positions I181and G182 and a substitution from Asn (N) to Phe (F) in position 193.

[0079] Other parent alpha-amylases contemplated include LE429, which isLE174 with an additional substitution in I201F. According to theinvention LE335 is the alpha-amylase, which in comparison to LE429 hasadditional substitutions in T49I+G107A; LE399 is LE335+G48A, i.e.,LE174, with G48A+T49I+G107A+I201F.

[0080] Altered Properties

[0081] The following section describes the relationship betweenmutations, which are present in a variant of the invention, anddesirable alterations in properties (relative to those of a parentTermamyl-like alpha-amylase), which may result therefrom.

[0082] As mentioned above the invention relates to a Termamyl-likealpha-amylase with altered properties.

[0083] Parent Termamyl-like alpha-amylaseS specifically contemplated inconnection with going through the specifically contemplated alteredproperties are the above mentioned parent Termamyl-like alpha-amylaseand parent hydrid Termamyl-like alpha-amylases.

[0084] The Bacllus licheniformis alpha-amylase (SEQ ID NO: 8) is used asthe starting point, but corresponding positions in, e.g., the SP722,BSG, BAN, AA560, SP690, KSM AP1378, #707 and other Termmayl-likealpha-amylases should be understood as disclosed and specificallycomtemplated too.

[0085] In an aspect the invention relates to variant with alteredproperties as mentioned above.

[0086] In the first aspect a variant of a parent Termamyl-likealpha-amylase, comprising an alteration at one or more positions (usingSEQ ID NO: 8 for the amino acid numbering) selected from the group of:

[0087] 5, 6, 36, 37, 38, 39, 42, 45, 47, 63, 66, 69, 70, 71, 72,74, 75,76, 79, 82, 83, 86, 87, 89, 93, 112, 113, 117, 120, 137, 213, 216, 220,223, 225, 226, 227, 229, 243, 245, 279, 282, 311, 321, 324, 352, 353,354, 357, 361, 362, 364, 368, 390, 395, 397, 399, 400, 401, 425, 451,452, 453, 466, 468, 470, 471,478,

[0088] wherein

[0089] (a) the alteration(s) are independently

[0090] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position,

[0091] (ii) a deletion of the amino acid which occupies the position, or

[0092] (iii) a substitution of the amino acid which occupies theposition with a different amino acid, (b) the variant has alpha-amylaseactivity and (c) each position corresponds to a position of the aminoadd sequence of the parent Termamyl-like alpha-amylase having the aminoacid sequence shown in SEQ ID NO: 8.

[0093] In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8the positions to be mutates are one or more of:

[0094] G5, T6, G36, I37, T38, A39, I42, A45, K47, D63, E66, Q69, K70,G71, T72, R74, T75, K76, T79, E82, L83, A86, I87, S89, R93, T112, E113,A117, V120, A137, K213, G216, A220, L223, L225, D226, G227, R229, D243,V245, F279, S282, T311, V321, V324, L352, T353, R354, G357, V361; F362,G364, G368, A390, A395, G397, Q399, H400, D401, A425, D451, I452, T453,G466, G468, F470, H471, S478.

[0095] In the first aspect the invention also relates to a variant of aparent Termamyl-like alpha-amylase, comprising a substitution at one ormore positions (using SEQ ID NO: 8 for the amino acid numbering)selected from the group of:

[0096] 7, 8, 9, 11, 12, 19, 21, 22, 25, 32, 40, 41, 46, 48, 55, 57, 58,60, 77, 95, 97, 98, 99, 100, 101, 102, 103, 105, 107, 115, 118, 135,139, 141, 143, 151, 159, 160, 161, 162, 163, 166, 175, 177, 183, 186,187, 192, 199, 200, 202, 203, 208, 212, 215, 219, 228, 230, 233, 236,238, 240, 241, 244, 248, 256, 258, 259, 260, 262, 270, 273, 274, 277,281, 283, 284, 285, 286, 287, 288, 289, 292, 295, 296, 304, 307, 312,313, 320, 322, 323, 325, 326, 478,327, 329, 331, 339, 343, 344, 346,347, 349, 350, 359, 360, 369, 377, 380, 387, 409, 410, 411, 412, 423,424, 426, 427, 428, 429, 430, 438,440, 441, 449, 462, 472, 477, 479,480, 481

[0097] wherein

[0098] (a) the variant has alpha-amylase activity and (b) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

[0099] In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8the positions to be mutated are one or more of:

[0100] L7, M8, Q9, F11, E12, G19, H21, W22, L25, L32, V40, W41, Y46,G48, G55, G57, A58, D60, Y77, I95, V97, Y98, G99, D100, V101, V102,I103, H105, G107, V115, V118, I135, T139, F141, F143, S151, H159, F160,D161, G162, T163, D166, Y175, F177, D183, V186, S187, N192, A199, D200,D202, Y203, V208, I212, W215, Y219, F228, L230, V233, I236, F238, F240,L241, W244, V248, M256, T258, V259, A260, Y262, L270, Y273, L274, T277,H281, V283, F284, D285, V286, P287, L288, H289, F292, A295, S296, M304,L307, V312, V313, S320, T322, F323, D325, N326, H327, T329, P331, V339,F343, K344, L346, A347, A349, F350, P359, Q360, T369, I377, L380, I387,V409, G410, W411, T412, G423, S478, L424, A426, L427, I428, T429, D430,M438, V440, G441, W449, I462, V472, V477, I479, Y480, V481

[0101] Specific substitutions contemplated are:

[0102] X7A,R,N,D,C,Q,E,G,H,K,M,P,S,Y,V;

[0103] X8C,M;

[0104] X9A,R,N,D,C,Q,G,H,M,P,S,T,W,Y,V;

[0105] X11A,N,D,C,Q,G,H,I,L,M,P,S,T,W,Y,V;

[0106] X12A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0107] X19A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0108] X21A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0109] X22A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0110] X25A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0111] X32A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0112] X40A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0113] X41A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0114] X46A,R,D,C,G,K,M,P,W,Y;

[0115] X48R,N,D,C,Q,E,G,H,K,M,F,P,W,Y;

[0116] X55A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0117] X57R,N,D,C,Q,E,G,H,K,M,P,W;

[0118] X58A,R,N,D,C,Q,E,G,H,K,M,S,T,W,Y;

[0119] X60A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0120] X77A,R,D,C,G,K,M,P,W,Y;

[0121] X95A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0122] X97A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0123] X98A,R,D,C,G,K,M,P,W,Y;

[0124] X99R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0125] X100A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[0126] X101A,N,C,Q,G,I,L,M,P,S,T,W,Y,V;

[0127] X102N,D,C,Q,E,H,I,L,M,F,P,W,Y,V;

[0128] X103A,N,D,C,Q,E,G,M,P,S,W,Y;

[0129] X105A,N,C,Q,G,H,I,L,M,P,S,T,Y,V;

[0130] X107R,N,D,Q,E,H,K,M,F,P,W,Y;

[0131] X115R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[0132] X118R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[0133] X135A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0134] X139A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0135] X141A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0136] X143A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[0137] X151A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;

[0138] X159A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;

[0139] X160A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0140] X161A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0141] X162A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0142] X163A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0143] X166A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0144] X175A,R,D,C,G,K,M,P,W,Y;

[0145] X177A,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;

[0146] X183A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0147] X186A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0148] X187A,R,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[0149] X192A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0150] X199R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0151] X200A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0152] X202A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0153] X203A,R,D,C,G,K,M,P;

[0154] X208A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[0155] X212A,N,C,Q,G,H,M,P,S,T,V;

[0156] X215A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0157] X219A,R,D,C,G,K,M,P,W,Y;

[0158] X228A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[0159] X230A,R,N,D,C,Q,E,G,L,M,P,S,T,W,Y,V;

[0160] X233R,N,C,Q,E,G,H,I,K,M,P,S,T,W,Y;

[0161] X236A,C,Q,G,H,I,M,P,S,T,V;

[0162] X238A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;

[0163] X240A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0164] X241A,N,C,Q,G,H,P,S,T,V;

[0165] X244A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0166] X248A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0167] X256C,M;

[0168] X258A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0169] X259A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

[0170] X260R,N,D,C,Q,E,H,I,L,K,M,F,P,T,W,Y,V;

[0171] X262A,R,D,C,G,K,M,P,W,Y;

[0172] X270A,N,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

[0173] X273A,R,D,C,G,K,M,P,Y;

[0174] X274A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0175] X277A,R,N,D,C,Q,E,G,H,K,M,P,S,W,Y,V;

[0176] X281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;

[0177] X283A,R,N,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[0178] X284A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,Y,V;

[0179] X285A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[0180] X286A,R,D,C,Q,E,G,H,l,K,M,F,P,S,T,W,Y,V; preferably

[0181] X286N,C,Q,I,L,M,P,T,V,Y,F;

[0182] X287R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0183] X288A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0184] X289A,R,N,D,C,Q,E;G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0185] X292A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0186] X295A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0187] X296A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0188] X304C,M;

[0189] X307A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0190] X312A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0191] X313A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0192] X320R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0193] X322R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[0194] X323A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[0195] X325A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0196] X326A,R,N,C,Q,E,G,M,P,S,T,W;

[0197] X327A,R,C,G,H,I,L,K,M,P,S,T,W,Y,V;

[0198] X329A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[0199] X331N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0200] X339A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0201] X343A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[0202] X344A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0203] X346A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0204] X347A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0205] X349R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0206] X350A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,Y,V;

[0207] X359R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0208] X360N,D,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0209] X369A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0210] X377A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0211] X380A,R,N,D,C,Q,E,G,H,K,P,S,W,Y,V;

[0212] X387A,R,N,D,C,Q,E,G,H,L,K,M,P,S,T,W,Y,V;

[0213] X409N,C,Q,E,G,H,M,P,S,T,W,Y,V;

[0214] X410A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0215] X411A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0216] X412R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0217] X423A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0218] X424A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0219] X426A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0220] X427A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;

[0221] X428A,N,D,Q,E,G,H,I,M,F,P,S,W,Y,V;

[0222] X429A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0223] X430A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0224] X438C,M;

[0225] X440R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0226] X441A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0227] X449A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0228] X462A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0229] X472A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0230] X477A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0231] X479A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0232] X480A,R,D,C,G,K,M,P,W,Y;

[0233] X481A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V

[0234] where each position corresponds to a position of the amino acidsequence of the parent Termamyl-like alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

[0235] Also according to the first aspect, the invention relates to avariant of a parent Termamyl-like alpha-amylase, comprising asubstitution at one or more positions (using SEQ ID NO: 8 for the aminoacid numbering) selected from the group of:

[0236] 1, 2, 3, 4, 13, 14, 16, 17, 18, 20, 23, 24, 26, 34, 35, 49, 50,51, 52, 53, 61, 62, 67, 68, 73, 84, 85, 88, 91, 92, 96, 106, 108, 114,116, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 136, 138, 145, 147, 148, 149, 150, 152, 153, 154, 155, 156,157, 158, 164, 165, 167, 168, 169, 170, 171, 172, 173, 176, 179, 180,181, 182, 184, 185, 188, 189,190, 191, 193, 196, 198, 204, 205, 206,209, 210, 211, 214, 217, 218, 221, 222, 234, 235, 237, 239, 242, 246,247, 249, 250, 251, 252, 253, 254, 255, 257, 261, 263, 265, 266, 267,268, 269, 271, 272, 275, 276, 278, 280, 290, 291, 293, 294, 297, 298,299, 300, 301, 302, 303, 305, 306, 308, 309, 310, 314, 315, 316, 317,318, 319, 328, 332, 333, 334, 335, 336, 337, 338, 340, 341, 342, 345,355, 358, 363, 370, 371, 373, 374, 375, 376, 378, 379, 381, 389, 393,394, 396, 398, 402, 403, 404, 405, 406, 407, 408, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 431, 432, 433, 434, 435, 436, 437, 439,442, 443, 444, 445, 446, 447, 448, 450, 454, 455, 456, 457, 458, 459,460, 461, 463, 464, 465, 467, 469, 473, 474, 475, 476, 482, 483

[0237] wherein

[0238] (a) the variant has alpha-amylase activity and (b) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

[0239] In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8the positions to be mutated are one or more of:

[0240] A1, N2, L3, N4, W13, Y14, P16, N17, D18, Q20, R23, R24, Q26, E34,H35, T49, S50, Q51, A52, D53, L61, Y62, F67, H68, V73, Q84, S85, K88,H91, S92, N96, K106, G108, D114, T116, E119, D121, P122, A123, D124,R125, N126, R127, V128, I129, S130, G131, E132, H133, L134, K136, W138,G145, G147, S148, T149, Y150, D152, F153, K154, W155, H156, W157, Y158,D164, W165, E167, S168, R169, K170, L171, N172, R173, K176, G179, K180,A181, W182, W184, E185, N188, E189, N190, G191, Y193, L196, Y198, D204,H205, P206, A209, A210, E211, R214, T217, W218, N221, E222, K234, H235,K237, S239, R242, N246, H247, R249, E250, K251, T252, G253, K254, E255,F257, E261, W263, N265, D266, L267, G268, A269, E271, N272, N275, K276,N278, N280, Y290, Q291, H293, A294, T297, Q298, G299, G300, G301, Y302,D303, R305, K306, L308, N309, G310, S314, K315, H316, P317, L318, K319,D328, G332, Q333, S334, L335, E336, S337, T338, Q340, T341, W342, P345,E355, Y358, Y363, K370, G371, S373, Q374, R375, E376, P378, A379, K381,K389, Q393, Y394, Y396, A398, Y402, F403, D404, H405, H406, D407, I408,R413, E414, G415, D416, S417, S418, V419, A420, N421, S422, G431, P432,G433, G434, A435, K436, R437, Y439, R442, Q443, N444, A445, G446, E447,T448, H450, G454, N455, R456, S457, E458, P459, V460, V461, N463, S464,E465, W467, E469, N473, G474, G475, S476, Q482, R483.

[0241] Specific substitutions contemplated are:

[0242] X1A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[0243] X2R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[0244] X3A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[0245] X4A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0246] X13R,N,D,C,Q,E,G,H,K,M,P,S,T,W;

[0247] X14A,R,D,C,G,K,M,P,W;

[0248] X16R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[0249] X17A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0250] X18A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0251] X20A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0252] X23A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

[0253] X24A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0254] X26A,D,C,E,G,H,I,L,M,F,P,S,T,W,V;

[0255] X34A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[0256] X35A,R,N,D,C,Q,E,G,H,K,M,F,P,S,T,W,Y,V;

[0257] X49A,C,G,H,P,T;

[0258] X50A,R,N,C,Q,E,G,H,K,M,F,P,S,W;

[0259] X51A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0260] X52A,R,D,C,Q,E,G,H,K,P;

[0261] X53A,D,C,G,H,K,M,P;

[0262] X61A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y;

[0263] X62A,R,D,C,G,K,M,P,Y;

[0264] X67A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[0265] X68A,R,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0266] X73A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

[0267] X84A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0268] X85A,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0269] X88A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0270] X91A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0271] X92A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0272] X96A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0273] X106A,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

[0274] X108R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0275] X114A,N,C,Q,E,G,H,K,F,P,S,T,W,Y;

[0276] X116A,R,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

[0277] X119A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0278] X121A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0279] X122R,N,Q,G,H,I,L,M,F,S,T,W,Y,V;

[0280] X123N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0281] X124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0282] X125R,N,Q,E,G,I,K,M,F,S,T,W,Y;

[0283] X126N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0284] X127A,R,N,D,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0285] X128A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,W,Y,V;

[0286] X129A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0287] X130A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0288] X131A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0289] X132R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y;

[0290] X133R,N,D,C,M,T,W,V;

[0291] X134A,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0292] X136A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0293] X138R,N,D,Q,E,G,I,K,M,P,S,T,W,V;

[0294] X145A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

[0295] X147A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[0296] X148A,R,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[0297] X149A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[0298] X150A,D,C,G,M,P,W,Y;

[0299] X152A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[0300] X153A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0301] X154A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0302] X155A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0303] X156A,C,Q,E,G,I,L,M,F,P,S,T,W,V;

[0304] X157R,I,L,M,F,P,S,T,W,Y,V;

[0305] X158R,M,P,W,Y;

[0306] X164R,I,L,M,F,P,S,T,W,Y,V;

[0307] X165A,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[0308] X167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0309] X168A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,V;

[0310] X169A,R,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

[0311] X170A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0312] X171A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

[0313] X172A,N,D,C,Q,E,G,I,L,M,F,P,T,W,Y,V;

[0314] X173A,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

[0315] X176A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0316] X179D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;

[0317] X180A,G,I,L,M,F,P,W,Y,V;

[0318] X181G;

[0319] X182A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0320] X184,I,L,M,F,P,W,Y,V;

[0321] X185R,I,L,M,F,P,S,T,W,Y,V;

[0322] X188A,R,N,Q,G,H,L,M,F,W,V;

[0323] X189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

[0324] X190N;

[0325] X191A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0326] X193A,R,G,M,P,W,Y;

[0327] X196A,N,Q,G,H,I,L,M,P,S,T,W,V;

[0328] X198A,R,G,M,P,W;

[0329] X204R,L,M,F,P,T,W,Y,V;

[0330] X205A,G,H,I,L,M,F,P,W,Y,V;

[0331] X206R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0332] X209R,P,S,W,Y;

[0333] X210A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[0334] X211E;

[0335] X214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;

[0336] X217A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y;

[0337] X218A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0338] X221A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0339] X222A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0340] X234A,D,C,G,H,I,K,M,F,P,S,T,W,Y,V;

[0341] X235A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0342] X237A,G,H,I,L,M,F,W,Y,V;

[0343] X239G,H,I,L,M,F,P,S,T,Y,V;

[0344] X242G,I,L,M,F,S,T,W,Y,V;

[0345] X246A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0346] X247R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;

[0347] X249A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0348] X250A,R,N,D,C,E,H,I,L,K,M,P,T,W,Y,V;

[0349] X251R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0350] X252A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0351] X253R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

[0352] X254A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0353] X255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;

[0354] X257A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0355] X261A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0356] X263A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[0357] X265C,Q,E,H,I,L,M,F,P,W;

[0358] X266A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0359] X267A,R,N,D,C,Q,E,G,H,K,P,S,T,W,Y,V;

[0360] X268A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0361] X269A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0362] X271A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0363] X272A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0364] X275A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0365] X276A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0366] X278A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0367] X280A,R,D,C,E,G,H,I,L,K,M,F,P,W,Y,V;

[0368] X290W,Y;

[0369] X291A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0370] X293A,R,N,G,I,L,M,P,S,T,W,V;

[0371] X294R,N,Q,G,H,I,L,M,F,P,S,T,W,Y;

[0372] X297A,G,H,I,L,M,F,P,W,Y,V;

[0373] X298G,H,I,L,M,F,P,S,T,W,Y,V;

[0374] X299A,G,H,M,P,S,T;

[0375] X300A,C,G,H,I,L,M,F,P,T,W,Y,V;

[0376] X301N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[0377] X302R,M,P,W,Y;

[0378] X303R,I,L,M,F,P,S,T,W,Y,V;

[0379] X305G,I,L,M,F,P,S,T,W,Y,V;

[0380] X306Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0381] X308A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0382] X309N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0383] X310A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[0384] X314A,D,C,E,G,I,L,M,F,P,W,Y,V;

[0385] X315A,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0386] X316A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;

[0387] X317R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0388] X318A,R,N,D,C,Q,E,G,H,I,K,P,S,W,Y,V;

[0389] X319A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0390] X328A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0391] X332A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0392] X333A,N,D,C,G,I,M,F,P,S,T,Y,V;

[0393] X334R,N,C,Q,E,G,H,K,M,F,P,S,W,Y;

[0394] X335R,D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;

[0395] X336A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0396] X337A,R,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0397] X338A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0398] X340I,L,M,F,P,S,T,W,Y,V;

[0399] X341A,R,D,C,G,H,I,L,K,M,F,W,Y,V;

[0400] X342R,I,L,M,F,P,S,T,W,Y,V;

[0401] X345R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[0402] X355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0403] X358A,R,D,C,G,K,M,P,W,Y;

[0404] X363A,R,D,C,G,K,M,P,W,Y;

[0405] X370A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0406] X371A,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0407] X373A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[0408] X374A,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[0409] X375A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,V;

[0410] X376A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0411] X378R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[0412] X379A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[0413] X381A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0414] X389A,D,C,G,H,M,F,P,S,T,W,Y,V;

[0415] X393A,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0416] X394A,R,D,C,G,K,M,P,W,Y;

[0417] X396A,R,D,C,G,K,M,P,W,Y;

[0418] X398A,R,N,D,C,Q,E,G,H,I,L,M,F,W,Y,V;

[0419] X402R,C,G,K,M,P,W,Y;

[0420] X403A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0421] X404R,I,L,M,F,P,S,T,W,Y,V;

[0422] X405R,G,H,I,L,M,F,P,W,Y,V;

[0423] X406A,R,G,H,I,M,F,P,Y,V;

[0424] X407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0425] X408A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;

[0426] X413A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0427] X414A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0428] X415A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0429] X416A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0430] X417A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[0431] X418A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0432] X419A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0433] X420A,N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[0434] X421A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[0435] X422A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0436] X431A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0437] X432G,H,I,L,M,F,P,S,T,W,Y,V;

[0438] X433A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0439] X434A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0440] X435Q,G,H,I,L,M,F,P,T,W,Y,V;

[0441] X436A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0442] X437A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0443] X439A,R,D,C,G,K,M,P,W,Y;

[0444] X442A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0445] X443A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0446] X444A,C,G,H,I,L,M,F,P,S,T,W,Y,V;

[0447] X445A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0448] X446A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0449] X447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;

[0450] X448A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0451] X450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[0452] X454A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0453] X455A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0454] X456A,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0455] X457A,R,N,D,C,Q,E,G,H,I,L,K,M,F,W,Y,V;

[0456] X458A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0457] X459R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y,V;

[0458] X460A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0459] X461A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

[0460] X463A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[0461] X464A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0462] X465A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0463] X467A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0464] X469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0465] X473N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0466] X474A,R,G,H,I,L,M,F,P,W,Y,V;

[0467] X475A,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0468] X476G,H,I,L,M,F,P,S,T,W,Y,V;

[0469] X482A,N,D,C,G,H,I,L,M,F,S,T,W,Y,V;

[0470] X483A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V

[0471] where each position corresponds to a position of the amino acidsequence of the parent Termamyl-like alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

[0472] Specifically contemplated according to the invention invention issubstitution at position 183 and/or 184 (using SEQ ID NO: 2 (SP690) forthe amino acid numbering) with any amino acid, i.e. any one of A, R, N,D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V.

[0473] Also in the first aspect the invention relates to a variant of aparent Termamyl-like alpha-amylase, comprising an alteration at one ormore positions (using SEQ ID NO: 8 for the amino acid numbering)selected from the group of: 1, 3, 4, 17, 18, 20, 23, 24, 28, 56, 61, 62,67, 68, 80, 81, 84, 85, 91, 92, 106, 110, 114, 119, 121, 122, 123, 124,125, 126, 127, 129, 131, 134, 136, 172, 185, 196, 206, 217, 218, 231,232, 235, 246, 247, 249, 251, 257, 278, 310, 316, 317, 328, 332, 355,358, 363, 367, 370, 373, 375, 376, 381, 382, 391, 396, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 439, 445, 446, 448, 450, 454, 455,458, 459, 460, 461, 463, 464, 465, 467

[0474] wherein

[0475] (a) the alteration(s) are independently

[0476] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position, or

[0477] (ii) a deletion of the amino acid which occupies the position,

[0478] (b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

[0479] In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8the positions to be mutated are one or more of:

[0480] A1, L3, N4, N17, D18, Q20, R23, R24, D28, Y56, L61, Y62, F67,H68, K80, G81, Q84, S85, H91, S92, K106, D110, D114, E119, D121, P122,A123, D124, R125, N126, R127,1129, G131, L134, K136, N172, E185, L196,P206, T217, W218, D231, A232, H235, N246, H247, R249, K251, F257, N278,G310, H316, P317, D328, G332, E355, Y358, Y363, Y367, K370, S373, R375,E376, K381, H382, R391, Y396, R413, E414, G415, D416, S417, S418, V419,A420, N421, S422, Y439, A445, G446, T448, H450, G454, N455, E458, P459,V460, V461, N463, S464, E465, W467.

[0481] Specific insertions/deletions contemplated are:

[0482] A1 insertion;

[0483] L3 insertion;

[0484] N4 insertion;

[0485] N17 insertion;

[0486] D18 insertion;

[0487] Q20 insertion;

[0488] R23 insertion;

[0489] R24 insertion;

[0490] D28 insertion;

[0491] Y56 insertion;

[0492] L61 insertion or deletion;

[0493] Y62 insertion;

[0494] F67 insertion or deletion;

[0495] H68 insertion;

[0496] K80 insertion or deletion;

[0497] G81 insertion or deletion;

[0498] Q84 insertion;

[0499] S85 insertion;

[0500] H91 insertion or deletion;

[0501] S92 insertion or deletion;

[0502] K106 insertion or deletion;

[0503] D110 insertion or deletion;

[0504] D114 deletion;

[0505] E119 insertion or deletion;

[0506] D121 insertion;

[0507] P122 insertion;

[0508] A123 insertion;

[0509] D124 insertion;

[0510] R125 insertion;

[0511] N126 insertion;

[0512] R127 insertion;

[0513] I129 insertion;

[0514] G131 insertion;

[0515] L134 insertion;

[0516] K136 insertion;

[0517] N172 insertion;

[0518] E185 insertion;

[0519] L196 insertion or deletion;

[0520] P206 insertion or deletion;

[0521] T217 insertion;

[0522] W218 insertion;

[0523] D231 insertion or deletion;

[0524] A232 insertion or deletion;

[0525] H235 insertion or deletion;

[0526] N246 insertion;

[0527] H247 insertion;

[0528] R249 insertion;

[0529] K251 insertion;

[0530] F257 insertion or deletion;

[0531] N278 insertion;

[0532] G310 insertion or deletion;

[0533] H316 insertion;

[0534] P317 insertion;

[0535] D328 insertion or deletion;

[0536] G332 insertion or deletion;

[0537] E355 insertion or deletion;

[0538] Y358 insertion;

[0539] Y363 insertion;

[0540] Y367 insertion;

[0541] K370 insertion;

[0542] S373 insertion;

[0543] R375 insertion;

[0544] E376 insertion;

[0545] K381 insertion;

[0546] H382 insertion;

[0547] R391 insertion or deletion;

[0548] Y396 insertion;

[0549] R413 insertion or deletion;

[0550] E414 insertion or deletion;

[0551] G415 insertion or deletion;

[0552] D416 insertion;

[0553] S417 insertion;

[0554] S418 insertion;

[0555] V419 insertion;

[0556] A420 insertion;

[0557] N421 insertion;

[0558] S422 insertion or deletion;

[0559] Y439 insertion;

[0560] A445 insertion or deletion;

[0561] G446 insertion or deletion;

[0562] T448 insertion or deletion;

[0563] H450 insertion;

[0564] G454 insertion or deletion;

[0565] N455 insertion;

[0566] E458 insertion;

[0567] P459 insertion;

[0568] V460 insertion;

[0569] V461 insertion;

[0570] N463 insertion;

[0571] S464 insertion;

[0572] E465 insertion;

[0573] W467 insertion

[0574] where each position corresponds to a position of the amino acidsequence of the parent Termamyl-like alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

[0575] Also in the first aspect the invention relates to a variant of aparent Termamyl-like alpha-amylase, comprising an alteration at one ormore positions (using SEQ ID NO: 8 for the amino acid numbering)selected from the group of:

[0576] 7, 8, 10, 11, 12, 15, 19, 21, 22, 25, 40, 41, 43, 44, 46, 55, 59,77, 78, 90, 95, 97, 98, 99, 100, 101, 102, 105, 109, 115, 118, 135, 139,141, 195, 208, 215, 219, 236, 238, 240, 244, 248, 256, 258, 259, 312,313, 320, 322, 323, 325, 326, 327, 330, 331, 348, 349, 350, 359, 360,365, 366, 369, 377, 384, 388, 423, 424, 438, 441, 449, 462, 479, 480,481

[0577] wherein

[0578] (a) the alteration(s) are independently

[0579] (i) an insertion of an amino acid downstream of the amino acidwhich occupies the position, or

[0580] (ii) a deletion of the amino acid which occupies the position,

[0581] (b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence shown in SEQID NO: 8.

[0582] In the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8the positions to be mutated are one or more of:

[0583] L7, M8, Y10, F11, E12, M15, G19, H21, W22, L25, V40, W41, P43,P44, Y46, G55, Y59, Y77, G78, L90, I95, V97, Y98, G99, D100, V101, V102,H105, A109, V115, V118, I135, T139, F141, Y195, V208, W215, Y219, I236,F238, F240, W244, V248, M256, T258, V259, V312, V313, S320, T322, F323,D325, N326, H327, Q330, P331, Y348, A349, F350, P359, Q360, D365, M366,T369, I377, I384, L388, G423, L424, M438, G441, W449, I462, I479, Y480,V481.

[0584] Specific insertions/deletions contemplated are:

[0585] L7 insertion or deletion;

[0586] M8 insertion;

[0587] Y10 insertion;

[0588] F11 insertion;

[0589] E12 insertion or deletion;

[0590] M15 insertion;

[0591] G19 insertion;

[0592] H21 insertion;

[0593] W22 insertion;

[0594] L25 insertion;

[0595] V40 insertion or deletion;

[0596] W41 insertion;

[0597] P43 insertion or deletion;

[0598] P44 insertion or deletion;

[0599] Y46 insertion;

[0600] G55 insertion;

[0601] Y59 insertion;

[0602] Y77 insertion;

[0603] G78 insertion or deletion;

[0604] L90 insertion or deletion;

[0605] I95 insertion;

[0606] V97 insertion;

[0607] Y98 insertion;

[0608] G99 insertion;

[0609] D100 insertion;

[0610] V101 insertion;

[0611] V102 insertion;

[0612] H105 insertion or deletion;

[0613] A109 insertion or deletion;

[0614] V115 insertion or deletion;

[0615] V118 insertion or deletion;

[0616] I135 insertion;

[0617] T139 insertion or deletion;

[0618] F141 insertion or deletion;

[0619] Y195 insertion;

[0620] V208 insertion or deletion;

[0621] W215 insertion;

[0622] Y219 insertion;

[0623] I236 insertion or deletion;

[0624] F238 insertion or deletion;

[0625] F240 insertion or deletion;

[0626] W244 insertion;

[0627] V248 insertion;

[0628] M256 insertion;

[0629] T258 insertion or deletion;

[0630] V259 insertion or deletion;

[0631] V312 insertion or deletion;

[0632] V313 insertion or deletion;

[0633] S320 insertion;

[0634] T322 insertion or deletion;

[0635] F323 insertion or deletion;

[0636] D325 insertion or deletion;

[0637] N326 insertion;

[0638] H327 insertion or deletion;

[0639] Q330 insertion or deletion;

[0640] P331 insertion or deletion;

[0641] Y348 insertion;

[0642] A349 insertion or deletion;

[0643] F350 insertion or deletion;

[0644] P359 insertion or deletion;

[0645] Q360 insertion;

[0646] D365 insertion or deletion;

[0647] M366 insertion;

[0648] T369 insertion;

[0649] I377 insertion;

[0650] I384 insertion or deletion;

[0651] L388 insertion or deletion;

[0652] G423 insertion or deletion;

[0653] L424 insertion or deletion;

[0654] M438 insertion;

[0655] G441 insertion or deletion;

[0656] W449 insertion;

[0657] I462 insertion;

[0658] I479 insertion or deletion;

[0659] Y480 insertion;

[0660] V481 insertion or deletion;

[0661] where each position corresponds to a position of the amino acidsequence of the parent Termamyl-like alpha-amylase having the amino acidsequence shown in SEQ ID NO: 8 (Bacillus licheniformis alpha-amylase).

[0662] Corresponding positions in other parent alpha-amylases can befound by alignment as described above and shown in the alignment in FIG.1.

[0663] Stability

[0664] In the context of the present invention, mutations (includingamino acid substitutions and deletion) of importance with respect toachieving altered stability, in particular improved stability (i.e.,higher or lower), at especially high temperatures (i.e., 70-120° C.)and/or extreme pH (i.e. low or high pH, i.e, pH 4-6 or pH 8-11,respectively), in particular at free (i.e., unbound, therefore insolution) calcium concentrations below 60 ppm, include any of themutations listed in the “Altered Properties” section. The stability maybe determined as described in the “Materials & Methods” section below.

[0665] Ca²⁺ Stability

[0666] Altered Ca²⁺ stability means the stability of the enzyme underCa²⁺ depletion has been improved, i.e., higher or lower stability. Inthe context of the present invention, mutations (including amino acidsubstitutions and deletions) of importance with respect to achievingaltered Ca²⁺ stability, in particular improved Ca²⁺ stability, i.e.,higher or lower stability, at especially high pH (i.e., pH 8-10.5)include any of the mutations listed in the in “Altered properties”section.

[0667] Specific Activity

[0668] In a further aspect of the present invention, important mutations(including amino acid substitutions and deletions) with respect toobtaining variants exhibiting altered specific activity, in particularincreased or decreased specific activity, especially at temperaturesfrom 10-60° C., preferably 20-50° C., especially 30-40° C., include anyof the mutations listed in the in “Altered properties” section. Thespecific activity may be determined as described in the “Material &Methods” section below.

[0669] Oxidation Stability

[0670] Variants of the invention may have altered oxidation stability,in particular higher oxidation stability, in comparison to the parentalpha-amylase. Increased oxidation stability is advantageous in, e.g.,detergent compositions and descresed oxidation stability may beadvantageous in composition for starch liquefaction. Oxidation stabilitymay be determined as described in the “Material & Methods” sectionbelow.

[0671] Altered pH Profile

[0672] Important positions and mutations with respect to obtainingvariants with altered pH profile, in particular improved activity atespecially high pH (i.e., pH 8-10.5) or low pH (i.e., pH 4-6) includemutations of amino residues located close to the active site residues.

[0673] Preferred specific mutations/substitutions are the ones listedabove in the section “Altered Properties” for the positions in question.Suitable assays are described in the “Materials & Methods” sectionbelow.

[0674] Wash Performance

[0675] Important positions and mutations with respect to obtainingvariants with improved wash performance at especially high pH (i.e., pH8.5-11) include the specific mutations/substitutions listed above in thesection “Altered Properties” for the positions in question. The washperformance may be tested as described below in the “Materials &Methods” section.

[0676] Increased pI

[0677] Substitutions Resulting in Higher pI

[0678] In an aspect the invention relates to a variant with a higher pithan the parent alpha-amylase. Such variants are suitable when adjustingthe pI to the washing conditions of various detergents. This means thatif the pI of the parent alpha-amylase is below the pH in the washingsolution the target is to increase the pI to the pH of the washingsolution. Such variant may be prepared by making the following kind ofsubstitutions:

[0679] 1) Substituting one or more of the below mentioned negativelycharged amino acid residue in a parent alpha-amylase with a positivelycharged amino acid residue.

[0680] 2) Substituting one or more of the below mentioned neutral aminoacid residue in a parent alpha-amylase with a positively charged aminoacid residue;

[0681] 3) Substituting one or more of the below mentioned negativelycharged amino acid residue in a parent alpha-amylase with a neutralamino acid residue;

[0682] 4) Substituting one or more of the below mentioned positivelycharged amino acid residue in a parent alpha-amylase with a morepositively charged amino acid residue;

[0683] Variants of the invention with increased pi in comparison to theparent alpha-amylase may have improved wash performance. Washperformance tests may be carried out as described in the “Materials &Method” section.

[0684] Thus, variants of the invention include (using SEQ ID NO: 8 forthe numbering):

[0685] G5R,K,H;

[0686] T6R,K,H;

[0687] G36R,K,H;

[0688] 137R,K,H;

[0689] T38R,K,H;

[0690] A39R,K,H;

[0691] I42R,K,H;

[0692] A45R,K,H;

[0693] D63A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0694] E66A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0695] Q69R,K,H;

[0696] G71R,K,H;

[0697] T72R,K,H;

[0698] T75R,K,H;

[0699] T79R,K,H;

[0700] E82A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0701] L83R,K,H;

[0702] A86R,K,H;

[0703] I87R,K,H;

[0704] S89R,K,H;

[0705] T112R,K,H;

[0706] E113A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0707] A117R,K,H;

[0708] V120R,K,H;

[0709] A137R,K,H;

[0710] G216R,K,H;

[0711] A220R,K,H;

[0712] L223R,K,H;

[0713] L225R,K,H;

[0714] D226A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0715] G227R,K,H;

[0716] D243A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0717] V245R,K,H;

[0718] F279R,K,H;

[0719] S282R,K,H;

[0720] T311R,K,H;

[0721] V321R,K,H;

[0722] V324R,K,H;

[0723] L352R,K,H;

[0724] T353R,K,H;

[0725] G357R,K,H;

[0726] V361R,K,H;

[0727] F362R,K,H;

[0728] G364R,K;H;

[0729] G368R,K,H;

[0730] A390R,K,H;

[0731] A395R,K,H;

[0732] G397R,K,H;

[0733] Q399R,K,H;

[0734] H400R,K,H;

[0735] D401A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0736] A425R,K,H;

[0737] D451A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0738] I452R,K,H;

[0739] T453R,K,H;

[0740] G466R,K,H;

[0741] G468R,K,H;

[0742] F470R,K,H;

[0743] S478R,K,H.

[0744] L7R,K,H;

[0745] Q9R,H,;

[0746] F11H;

[0747] E12A,R,N,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0748] G19R,K,H;

[0749] W22R,K,H;

[0750] L25R,K,H;

[0751] L32R,K,H;

[0752] V40R,K,H;

[0753] W41 R,K,H;

[0754] Y46R,K;

[0755] G48R,K,H;

[0756] G55R,K,H;

[0757] G57R,K,H;

[0758] A58R,K,H;

[0759] D60A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0760] Y77R,K;

[0761] 195R,K,H;

[0762] V97R,K,H;

[0763] Y98R,K,;

[0764] G99R,K,H;

[0765] D100A,R,C,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

[0766] V102H;

[0767] G107R,K,H;

[0768] V115R,K,H;

[0769] V118R,K,H;

[0770] I135R,K,H;

[0771] T139R,K,H;

[0772] F141R,K,H;

[0773] F143R,K,H;

[0774] S151R,K,H;

[0775] F160R,K,H;

[0776] D161R,K,H;

[0777] G162R,K,H;

[0778] T163R,K,H;

[0779] D166A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0780] Y175R,K,;

[0781] F177K,H;

[0782] D183A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0783] V186R,K,H;

[0784] S187R,K,H;

[0785] N192R,K,H;

[0786] A199R,K,H;

[0787] D200A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0788] D202A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0789] Y203R,K;

[0790] V208R,K,H;

[0791] I212H;

[0792] W215R,K,H;

[0793] Y219R,K;

[0794] F228R,K,H;

[0795] L230R;

[0796] V233R,K,H;

[0797] I236H;

[0798] F238R,K,H;

[0799] F240R,K,H;

[0800] L241H;

[0801] W244R,K,H;

[0802] V248R,K,H;

[0803] T258R,K,H;

[0804] V259R,K,H;

[0805] A260R,K,H;

[0806] Y262R,K;

[0807] Y273R,K;

[0808] L274R,K,H;

[0809] T277R,K,H;

[0810] H281R,K;

[0811] V283R,K;

[0812] F284RK;

[0813] D285A,R,C,Q,G,H,I,K,M,F,P,S,T,W,Y,V;

[0814] V286R,K,H;

[0815] P287R,H;

[0816] I288R,K,H;

[0817] H289R,K;

[0818] F292R,K,H;

[0819] A295R,K,H;

[0820] S296R,K,H;

[0821] L307R,K,H;

[0822] V312H;

[0823] V313R,K,H;

[0824] S320R,K,H;

[0825] T322R,K,H;

[0826] F323R,K;

[0827] D325A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0828] N326R;

[0829] T329R,K,H;

[0830] P331H;

[0831] V339R,H,K;

[0832] F343R,H,K;

[0833] K344H;

[0834] L346R,K,H;

[0835] A347R,K,H;

[0836] A349R,K,H;

[0837] F350R,K,H;

[0838] P359R,K,H;

[0839] Q360H;

[0840] T369R,K,H;

[0841] I377R,K,H;

[0842] L380RK,H;

[0843] I387R,K,H;

[0844] V409H;

[0845] G410R,K,H;

[0846] W411R,K,H;

[0847] T412R,K,H;

[0848] G423R,K,H;

[0849] L424R,K,H;

[0850] A426R,K,H;

[0851] L427R,K,H;

[0852] I428H;

[0853] T429R,K,H;

[0854] D430A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0855] V440R,K,H;

[0856] G441R,K,H;

[0857] W449R,K,H;

[0858] I462R,K,H;

[0859] V472R,K,H;

[0860] V477R,K,H;

[0861] I479R,K,H;

[0862] Y480R,K;

[0863] V481R,K,H.

[0864] A1R,K,H;

[0865] N2R,K,H;

[0866] L3R,K,H;

[0867] N4R,K,H;

[0868] W13R,K,H;

[0869] Y14R,K;

[0870] P16R,K,H;

[0871] N17R,K,H;

[0872] D18A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0873] Q20R,K,H;

[0874] R23H;

[0875] R24K,H;

[0876] Q26H;

[0877] E34A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

[0878] T49H;

[0879] S50R,K,H;

[0880] Q51H;

[0881] A52R,K,H;

[0882] D53A,C,G,H,K,M,P;

[0883] L61R,K,H;

[0884] Y62R,K;

[0885] F67R,K,H;

[0886] V73R,K,H;

[0887] Q84R,K,H;

[0888] S85R,K,H;

[0889] K88H;

[0890] S92R,H;

[0891] N96R,K,H;

[0892] K106H;

[0893] G108R,K,H;

[0894] D114A,N,C,Q,G,H,K,F,P,S,T,W,Y;

[0895] T116R,H;

[0896] E119A,R,N,D,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0897] D121A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0898] P122R,H;

[0899] A123H;

[0900] D124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0901] R125K;

[0902] N126H;

[0903] R127K,H;

[0904] V128R,K,H;

[0905] I129R,K,H;

[0906] S130R,K,H;

[0907] G131R,K,H;

[0908] E132R,N,D,C,Q,G,H,I,L,K,M,F,S,W,Y;

[0909] L134K,H;

[0910] K136A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[0911] W138R,K;

[0912] G145R,K,H;

[0913] G147R,K,H;

[0914] S148R,K,H;

[0915] T149R,K,H;

[0916] D152A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

[0917] F153R,K,H;

[0918] K154H;

[0919] W155R,K,H;

[0920] W157R;

[0921] Y158R;

[0922] D164R,I,L,M,F,P,S,T,W,Y,V;

[0923] W165K,H;

[0924] E167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0925] S168R,K,H;

[0926] R169H;

[0927] K170H;

[0928] L171R,K,H;

[0929] R173H;

[0930] K176H;

[0931] G179K,H;

[0932] W182R,K,H;

[0933] E185R,I,L,M,F,P,S,T,W,Y,V;

[0934] N188R,H;

[0935] E189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

[0936] G191R,H;

[0937] Y193R;

[0938] L196H;

[0939] Y198R;

[0940] D204R,L,M,F,P,T,W,Y,V;

[0941] P206R,K,H;

[0942] A209R;

[0943] A210R,K,H;

[0944] T217R,H;

[0945] W218R,K,H;

[0946] N221R,K,H;

[0947] E222R,K,H;

[0948] K234H;

[0949] K237H;

[0950] S239H;

[0951] N246R,K,H;

[0952] R249H;

[0953] E250A,R,N,D,C,H,I,L,K,M,P,T,W,Y,V;

[0954] K251H;

[0955] T252R,K,H;

[0956] G253R,K,H;

[0957] K254H;

[0958] E255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;

[0959] F257R,K,H;

[0960] E261A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0961] W263R,K;

[0962] N265H;

[0963] D266A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0964] L267R,K,H;

[0965] G268R,K,H;

[0966] A269H;

[0967] E271A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0968] N272R,K,H;

[0969] N275R,K,H;

[0970] K276H;

[0971] N278R,K,H;

[0972] N280R,K,H;

[0973] Q291R,H;

[0974] A294R,H;

[0975] T297H;

[0976] Q298H;

[0977] G299H;

[0978] G300H;

[0979] G301H;

[0980] Y302R;

[0981] D303R,I,L,M,F,P,S,T,W,Y,V;

[0982] K306H;

[0983] L308R,K,H;

[0984] N309H;

[0985] G310R,K,H;

[0986] K315H;

[0987] P317R,K,H;

[0988] L318R,K,H;

[0989] K319H;

[0990] D328A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[0991] G332R,K,H;

[0992] S334R,K,H;

[0993] L335R,K,H;

[0994] E336A,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[0995] S337R,H;

[0996] T338R,K,H;

[0997] T341R,K,H;

[0998] W342R;

[0999] P345R,K,H;

[1000] E355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[1001] Y358R,K;

[1002] Y363R,K;

[1003] K370H;

[1004] G371K,H;

[1005] S373R,K,H;

[1006] Q374K,H;

[1007] R375H;

[1008] E376A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[1009] P378R,K,H;

[1010] A379R,K,H;

[1011] K381H;

[1012] K389H;

[1013] Q393K,H;

[1014] Y394R,K;

[1015] Y396R,M;

[1016] A398R,H;

[1017] Y402R,K;

[1018] F403R,H;

[1019] D404R,I,L,M,F,P,S,T,W,Y,V;

[1020] H406R;

[1021] D407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[1022] I408R,K,H;

[1023] R413K,H;

[1024] E414A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[1025] G415R,K,H;

[1026] D416A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[1027] S417R,K,H;

[1028] S418R,K,H;

[1029] V419R,H;

[1030] A420K,H;

[1031] N421R,K,H;

[1032] S422R,K,H;

[1033] G431R,H;

[1034] P432H;

[1035] G433R,H;

[1036] G434R;

[1037] A435H;

[1038] K436H;

[1039] R437K,H;

[1040] Y439R,K;

[1041] R442H;

[1042] Q443R,H;

[1043] N444H;

[1044] A445R,K,H;

[1045] G446R,K,H;

[1046] E447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;

[1047] T448R,K,H;

[1048] G454R,K,H;

[1049] N455R,K,H;

[1050] R456H;

[1051] S457R,K,H;

[1052] E458A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[1053] P459R,K,H;

[1054] V460R,K,H;

[1055] V461R,H;

[1056] N463R,K,H;

[1057] S464R,K,H;

[1058] E465A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[1059] W467R,K,H;

[1060] E469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[1061] N473H;

[1062] G474R,H;

[1063] G475H;

[1064] S476H;

[1065] Q482H;

[1066] R483H.

[1067] Decreased pI

[1068] Substitutions Resulting in Lower pI

[1069] Variants of the invention with decreased pi in comparison to theparent alpha-amylase may have improved liquefying effect. This meansthat the pI of the parent amylase should be adjusted to the pHconditions in the liquefying process in question. Normally the pH duringliquefaction lies in the range of 4-7, such as between pH 4.5-6.5. Anexample of a liquefaction process is descripted below in the section“Liquefaction”. Improved liquefying effect may be carried out asdescribed in the “Materials & Method” section.

[1070] Alternatively, variants with decreased pI may advantageously beused in detergent. If the pI of the parent alpha-amylase is above the pHin the washing solution the target is to decrease the pI to the pH ofthe washing solution. Such variant may be prepared by making thefollowing kind of substitutions:

[1071] 1) Substituting one or more of the below mentioned positivelycharged amino acid residue in a parent alpha-amylase with a neutralamino acid residue.

[1072] 2) Substituting one or more of the below mentioned neutral aminoacid residue in a parent alpha-amylase with a negatively charged aminoacid residue.

[1073] 3) Substituting one or more of the below mentioned positivelycharged amino acid residue with a negatively charged amino acid residue.

[1074] 4) Substituting one or more of the below mentioned negativelycharged amino acid residue with a more negatively charged amino acidresidue.

[1075] Thus, variants of the invention include (using SEQ ID NO: 8 forthe numbering):

[1076] G5D,E;

[1077] T6D,E;

[1078] G36D,E;

[1079] I37D,E;

[1080] T38D,E;

[1081] A39D,E;

[1082] I42D,E;

[1083] A45D,E;

[1084] K47A,R,N,D,C,Q,E,G,H,,M,F,P,S,T,W,Y,V;

[1085] D63E;

[1086] Q69D,E;

[1087] K70A,N,D,C,Q,E,G,I,M,F,P,S,T,W,Y,V;

[1088] G71D,E;

[1089] T72D,E;

[1090] R74A,N,D,C,Q,E,G,I,M,F,P,S,T,W,Y,V;

[1091] T75D,E;

[1092] K76A,N,D,C,Q,E,G,L,M,F,P,S,T,W,Y,V;

[1093] T79D,E;

[1094] L83D,E;

[1095] A86D,E;

[1096] 187D,E;

[1097] S89D,E;

[1098] R93A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1099] T112D,E;

[1100] A117D,E;

[1101] V120D,E;

[1102] A137D,E;

[1103] K213A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1104] G216D,E;

[1105] A220D,E;

[1106] L223D,E;

[1107] L225D,E;

[1108] D226E;

[1109] G227D,E;

[1110] R229A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1111] D243E;

[1112] V245D,E;

[1113] F279D,E;

[1114] S282D,E;

[1115] T311D,E;

[1116] V321D,E;

[1117] V324D,E;

[1118] L352D,E;

[1119] T353D,E;

[1120] R354A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1121] G357D,E;

[1122] V361D,E;

[1123] F362D,E;

[1124] G364D,E;

[1125] G368D,E;

[1126] A390D,E;

[1127] A395D,E;

[1128] G397D,E;

[1129] Q399D,E;

[1130] H400A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1131] D401E;

[1132] A425D,E;

[1133] D451E;

[1134] I452D,E;

[1135] T453D,E;

[1136] G466D,E;

[1137] G468D,E;

[1138] F470D,E;

[1139] H471A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1140] S478D,E.

[1141] L7D,E;

[1142] Q9D;

[1143] F11

[1144] D;

[1145] G19D,E;

[1146] H21R,D,E,K;

[1147] W22D,E;

[1148] L25D,E;

[1149] L32D,E;

[1150] V40D,E;

[1151] W41D,E;

[1152] Y46D;

[1153] G48D,E;

[1154] G55D,E;

[1155] G57D,E;

[1156] A58D,E;

[1157] D60E;

[1158] Y77D;

[1159] I95D,E;

[1160] V97D,E;

[1161] Y98D;

[1162] G99D,E;

[1163] D100E;

[1164] V102D,E;

[1165] I103A,D,E;

[1166] H105A,N,C,Q,G,I,L,M,P,S,T,Y,V;

[1167] G107D,E;

[1168] V115D,E;

[1169] V118E;

[1170] I135D,E;

[1171] T139D,E;

[1172] F141D,E;

[1173] F143AD,E;

[1174] S151D,E;

[1175] H159A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[1176] F160D,E;

[1177] D161E;

[1178] G162D,E;

[1179] T163D,E;

[1180] Y175D;

[1181] F177D,E;

[1182] D183E;

[1183] V186D,E;

[1184] S187E;

[1185] N192D,E;

[1186] A199D,E;

[1187] D200E;

[1188] D202E;

[1189] Y203D;

[1190] V208D,E;

[1191] W215D,E;

[1192] Y219D;

[1193] F228D,E;

[1194] L230D,E;

[1195] V233E;

[1196] F238D,E;

[1197] F240D,E;

[1198] W244D,E;

[1199] V248D,E;

[1200] T258D,E;

[1201] V259D,E;

[1202] A260D,E;

[1203] Y262D;

[1204] Y273D;

[1205] L274D,E;

[1206] T277D,E;

[1207] H281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;

[1208] V283E;

[1209] F284D,E;

[1210] D285E;

[1211] V286D,E;

[1212] P287D,E;

[1213] L288D,E;

[1214] H289A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[1215] F292D,E;

[1216] A295D,E;

[1217] S296D,E;

[1218] L307D,E;

[1219] V313D,E;

[1220] S320D,E;

[1221] T322D,E;

[1222] F323D,E;

[1223] D325E;

[1224] N326E;

[1225] H327A,R,C,G,I,L,K,M,P,S,T,W,Y,V;

[1226] T329D,E;

[1227] P331D,E;

[1228] V339E;

[1229] F343D,E;

[1230] K344A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1231] L346D,E;

[1232] A347D,E;

[1233] A349D,E;

[1234] P359D,E;

[1235] Q360D;

[1236] T369D,E;

[1237] I377D,E;

[1238] L380D,E;

[1239] I387D,E;

[1240] V409E;

[1241] G410D,E;

[1242] W411D,E;

[1243] T412E;

[1244] G423D,E;

[1245] L424D,E;

[1246] A426D,E;

[1247] L427D,E;

[1248] I428D,E;

[1249] T429D,E;

[1250] D430E;

[1251] V440D,E;

[1252] G441D,E;

[1253] W449D,E;

[1254] I462D,E;

[1255] V472D,E;

[1256] V477D,E;

[1257] I479D,E;

[1258] Y480D;

[1259] V481D,E.

[1260] A1D,E;

[1261] N2D,E;

[1262] L3D,E;

[1263] N4D,E;

[1264] W13D,E;

[1265] Y14D;

[1266] P16D,E;

[1267] N17D,E;

[1268] D18E;

[1269] Q20D,E;

[1270] R23D,E;

[1271] R24D,E;

[1272] Q26D,E;

[1273] H35A,R,N,D,C,Q,E,G,K,M,F,P,S,T,W,Y,V;

[1274] S50E;

[1275] Q51D,E;

[1276] A52D,E;

[1277] L61D,E;

[1278] Y62D;

[1279] F67D,E;

[1280] H68A,R,D,C,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[1281] V73D,E;

[1282] Q84D;

[1283] S85E;

[1284] K88A,R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

[1285] H91A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[1286] S92D,E;

[1287] N96D,E;

[1288] K106A,N,D,C,Q,E,G,I,L,M,P,S,T,Y,V;

[1289] G108D,E;

[1290] D114E;

[1291] T116D,E;

[1292] D121E;

[1293] A123D,E;

[1294] R125N,Q,E,G,I,M,F,S,T,W,Y;

[1295] R127D,E;

[1296] V128D;

[1297] I129D,E;

[1298] S130D,E;

[1299] G131D;

[1300] H133R,N,D,C,M,T,W,V;

[1301] L134D,E;

[1302] K136A,R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

[1303] W138D,E;

[1304] G145D,E;

[1305] G147D,E;

[1306] S148D,E;

[1307] T149D,E;

[1308] Y150D;

[1309] D152E;

[1310] F153D,E;

[1311] K154A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1312] W155D,E;

[1313] H156A,C,Q,E,G,I,L,M,F,P,S,T,W,V;

[1314] W165D,E;

[1315] S168D,E;

[1316] R169D,E;

[1317] K170A,R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

[1318] L171D,E;

[1319] N172D,E;

[1320] R173A,N,D,C,Q,E,G,M,P,S,W,Y,V;

[1321] K176A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1322] G179D,E;

[1323] K180A,G,I,L,M,F,P,W,Y,V;

[1324] W182D,E;

[1325] P206D,E;

[1326] A210D,E;

[1327] R214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;

[1328] T217D;

[1329] W218D,E;

[1330] N221D,E;

[1331] K234A,D,C,G,I,M,F,P,S,T,W,Y,V;

[1332] H235A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[1333] K237A,G,I,L,M,F,W,Y,V;

[1334] R242G,I,L,M,F,S,T,W,Y,V;

[1335] N246D,E;

[1336] H247R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[1337] R249A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1338] K251R,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

[1339] T252D,E;

[1340] G253E;

[1341] K254A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1342] F257D,E;

[1343] W263D,E;

[1344] N265E;

[1345] D266E;

[1346] L267D,E;

[1347] G268D,E;

[1348] N272D,E;

[1349] N275D,E;

[1350] K276A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1351] N278D,E;

[1352] N280D,E;

[1353] H293A,R,N,G,I,L,M,P,S,T,W,V;

[1354] R305G,I,L,M,F,P,S,T,W,Y,V;

[1355] K306Q,G,I,L,M,F,P,S,T,W,Y,V;

[1356] L308D,E;

[1357] G310E;

[1358] S314D,E;

[1359] K315A,N,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1360] H316A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[1361] P317D,E;

[1362] L318D,E;

[1363] K319A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1364] D328E;

[1365] G332E;

[1366] Q333D;

[1367] S334E;

[1368] L335D,E;

[1369] S337E;

[1370] T341D;

[1371] P345D,E;

[1372] Y358D;

[1373] Y363D;

[1374] K370A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1375] G371E;

[1376] S373D,E;

[1377] Q374D,E;

[1378] R375A,N,D,C,Q,G,I,L,M,F,P,S,T,W,V;

[1379] P378D,E;

[1380] A379D,E;

[1381] K381A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1382] K389A,D,C,G,M,F,P,S,T,W,Y,V;

[1383] Q393E;

[1384] Y394D;

[1385] Y396D;

[1386] A398D,E;

[1387] H405R,G,I,L,M,F,P,W,Y,V;

[1388] H406A,R,G,I,M,F,P,Y,V;

[1389] I408D,E;

[1390] R413A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1391] G415D,E;

[1392] D416E;

[1393] S417D,E;

[1394] S418D,E;

[1395] V419D,E;

[1396] A420D,E;

[1397] N421D,E;

[1398] S422D,E;

[1399] K436A,R,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1400] R437A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V;

[1401] Y439D;

[1402] R442A,N,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

[1403] Q443D,E;

[1404] A445D,E;

[1405] G446D,E;

[1406] T448D,E,;

[1407] H450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[1408] G454D,E;

[1409] R456A,D,C,E,G,I,L,M,F,P,S,T,W,Y,V;

[1410] S457D,E;

[1411] P459D,E;

[1412] V460D,E;

[1413] V461D,E;

[1414] N463D,E;

[1415] S464D,E;

[1416] W467D,E;

[1417] Q482D;

[1418] R483A,N,D,C,Q,E,G,I,L,M,F,P,S,T,W,Y,V.

[1419] Reduced Sensitivity to Anionic Surfactants

[1420] Substitutions Resulting in a More Hydrophilic Amino Acid Residue

[1421] In an aspect, the invention relates to providing alpha-amylasevariants with reduced sensitivity (or improved stability againstdenaturation) to anionic surfactants (in particular linear alkylsulphonates (LAS)). These variants are provided by substituting,deleting or inserting an amino acid residue in the parent alpha-amylaseas indicated below with a more hydrophilic amino acid residue. Suchvariants may be prepared by:

[1422] 1) Substituting one or more of the below mentioned positivelycharged amino acid residue in a parent alpha-amylase with a hydrophilicamino acid residue.

[1423] 2) Substituting one or more of the below mentioned hydrophobicamino acid residue in a parent alpha-amylase with a hydrophilic aminoacid residue

[1424] 3) Substituting one or more of the below mentioned positivelycharged amino acid residue in a parent alpha-amylase with a netral ornegatively charged amino acid residue.

[1425] The anionic surfactants (in particular linear alkyl sulphonates(LAS)) sensitivity (in detergent) may be tested as described in the“Materials & Methods” section.

[1426] Variants of the invention with reduced sensitivity to anionicsurfactants, in particular linear alkyl sulphonates (LAS), include(using the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8numbering):

[1427] G5N,C,Q,S,T;

[1428] T6N,C,Q,S;

[1429] G36N,C,Q,S,T;

[1430] I37N,C,Q,S,T;

[1431] T38N,C,Q,S;

[1432] A39N,C,Q,S,T;

[1433] I42N,C,Q,S,T;

[1434] A45N,C,Q,S,T;

[1435] K47N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1436] Q69N;

[1437] K70N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1438] G71N,D,C,Q,S,T;

[1439] T72N,C,Q,S;

[1440] R74N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1441] T75N,C,Q,S;

[1442] K76N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1443] T79N,C,Q,S;

[1444] L83N,C,Q,S,T;

[1445] A86N,C,Q,S,T;

[1446] 187N,C,Q,S,T;

[1447] S89N,C,Q;

[1448] R93N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1449] T112N,C,Q,S;

[1450] A117N,C,Q,S,T;

[1451] V120N,C,Q,S,T;

[1452] A137N,C,Q,S,T;

[1453] K213N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1454] G216N,C,Q,S,T;

[1455] A220N,C,Q,S,T;

[1456] L223N,C,Q,S,T;

[1457] L225N,C,Q,S,T;

[1458] G227N,C,Q,S,T;

[1459] R229N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1460] V245N,C,Q,S,T;

[1461] F279N,C,Q,S,T;

[1462] S282N,C,Q;

[1463] T311N,,C,Q,S;

[1464] V321N,C,Q,S,T;

[1465] V324N,C,Q,S,T;

[1466] L352N,C,Q,S,T;

[1467] T353N,C,Q,S;

[1468] R354N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1469] G357N,C,Q,S,T;

[1470] V361N,C,Q,S,T;

[1471] F362N,C,Q,S,T;

[1472] G364N,C,Q,S,T;

[1473] G368N,C,Q,S,T;

[1474] A390N,C,Q,S,T;

[1475] A395N,C,Q,S,T;

[1476] G397N,C,Q,S,T;

[1477] Q399N;

[1478] H400N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1479] A425N,C,Q,S,T;

[1480] I452N,C,Q,S,T;

[1481] T453N,C,Q,S;

[1482] G466N,C,Q,S,T;

[1483] G468N,C,Q,S,T;

[1484] F470N,C,Q,S,T;

[1485] H471N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1486] S478N,C,Q.

[1487] L7N,C,Q,S;

[1488] M8C;

[1489] Q9N;

[1490] F11N,C,Q,S,T;

[1491] G19N,C,Q,S,T;

[1492] H21N,C,Q,S,T, A,I,L,M,F,P,W,Y,V, D,E;

[1493] W22N,C,Q,S,T;

[1494] L25N,C,Q,S,T;

[1495] L32N,C,Q,S,T;

[1496] V40N,C,Q,S,T;

[1497] W41N,C,Q,S,T;

[1498] Y46C;

[1499] G48N,C,Q;

[1500] G55N,C,Q,S,T;

[1501] G57N,C,Q;

[1502] A58N,C,Q,S,T;

[1503] Y77C;

[1504] I95N,C,Q,S,T;

[1505] V97N,C,Q,S,T;

[1506] Y98C;

[1507] G99N,C,Q,S,T;

[1508] V101N,C,Q,S,T;

[1509] V102N,C,Q;

[1510] I103N,C,Q,S;

[1511] H105N,C,Q,S,T, A,I,L,M,P,Y,V,;

[1512] G107N,Q;

[1513] V15N,C,Q,S,T;

[1514] V18N,C,Q,S,T;

[1515] I135N,C,Q,S,T;

[1516] T139N,C,Q,S;

[1517] F141N,C,Q,S,T;

[1518] F143N,C,Q,S,T;

[1519] S151N,C,Q;

[1520] H159N,C,Q,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1521] F160N,C,Q,S,T;

[1522] G162N,C,Q,S,T;

[1523] T163N,C,Q,S;

[1524] Y175C;

[1525] F177N,C,Q,S,T;

[1526] V186N,C,Q,S,T;

[1527] S187C,Q;

[1528] A199N,C,Q,S,T;

[1529] Y203C;

[1530] V208N,C,Q,S,T;

[1531] I212N,C,Q,S,T;

[1532] W215N,C,Q,S,T;

[1533] Y219C;

[1534] F228N,C,Q,S,T;

[1535] L230N,C,Q,S,T;

[1536] V233N,C,Q,S,T;

[1537] I236C,Q,S,T;

[1538] F238N,C,Q,S,T;

[1539] F240N,C,Q,S,T;

[1540] L241N,C,Q,S,T;

[1541] W244N,C,Q,S,T;

[1542] V248N,C,Q,S,T;

[1543] M256C;

[1544] T258N,C,Q,S;

[1545] V259N,C,Q,S,T;

[1546] A260N,C,Q,T;

[1547] Y262C;

[1548] L270N,C,Q,S,T;

[1549] Y273C;

[1550] L274N,C,Q,S,T;

[1551] T277N,C,Q,S;

[1552] H281 N,C,Q,S,T, A,M,P,W,Y,V, D,E;

[1553] V283N,C,Q,S,T;

[1554] F284N,C,Q,S,T;

[1555] V286C,Q,S,T;

[1556] P287N,C,Q,S,T;

[1557] L288N,C,Q,S,T;

[1558] H289N,C,Q,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1559] F292N,C,Q,S,T;

[1560] A295N,C,Q,S,T;

[1561] S296N,C,Q,T;

[1562] M304C;

[1563] L307N,C,Q,S,T;

[1564] V312N,C,Q,S,T;

[1565] V313N,C,Q,S,T;

[1566] S320N,C,Q;

[1567] T322N,C,Q,S;

[1568] F323N,C,Q,S,T;

[1569] H327C,S,T, A,I,L,M,P,W,Y,V;

[1570] T329N,C,Q;

[1571] P331N,C,Q,S,T;

[1572] V339N,C,Q,S,T;

[1573] F343N,C,Q,S,T;

[1574] K344N,C,Q,S,T;

[1575] L346N,Q,S,T;

[1576] A347N,C,Q,S,T;

[1577] A349N,Q,S,T;

[1578] F350N,C,Q,S,T;

[1579] P359N,C,Q,S,T;

[1580] Q360N;

[1581] T369N,C,Q,S;

[1582] I377N,C,Q,S,T;

[1583] L380N,C,Q,S;

[1584] I387N,C,Q,S,T;

[1585] V409N,C,Q,S,T;

[1586] G410N,C,Q,S,T;

[1587] W411N,C,Q,S,T;

[1588] T412N,C,Q,S;

[1589] G423N,C,Q,S,T;

[1590] L424N,C,Q,S,T;

[1591] A426N,C,Q,S,T;

[1592] L427N,C,Q,S,T;

[1593] I428N,Q,S;

[1594] T429N,C,Q,S;

[1595] M438C;

[1596] V440N,C,Q,S,T;

[1597] G441N,C,Q,S,T;

[1598] W449N,C,Q,S,T;

[1599] I462N,C,Q,S,T;

[1600] V472N,C,Q,S,T;

[1601] V477N,C,Q,S,T;

[1602] I479N,Q,S;

[1603] Y480C;

[1604] V481N,C,Q,S,T.

[1605] A1N,C,Q,S,T;

[1606] L3N,C,Q,S,T;

[1607] N4C,Q,S,T;

[1608] W13N,C,Q,S,T;

[1609] Y14C;

[1610] P16N,C,Q,S,T;

[1611] Q20N;

[1612] R23N,C,Q,S, A,I,L,M,F,P,W,Y,V,D,E;

[1613] R24N,C,Q,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1614] H35N,C,Q,S,T, A,M,F,P,W,Y,V,D,E;

[1615] T49C;

[1616] S50N,C,Q;

[1617] Q51N;

[1618] A52C,Q;

[1619] L61N,C,Q,S,T;

[1620] Y62C;

[1621] F67N,C,Q,S,T;

[1622] H68C,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1623] V73N,C,Q,S,T;

[1624] Q84N;

[1625] S85N,C,T;

[1626] K88N,C,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1627] H91N,C,Q,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1628] S92N,C,Q;

[1629] K106N,C,Q,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1630] G108N,C,Q,S,T;

[1631] T116C,Q,S;

[1632] E119N,Q,S,T;

[1633] P122N,Q,S,T;

[1634] A123N,C,Q,S,T;

[1635] R125N,Q,S,T, I,M,F,W,Y,E;

[1636] R127N,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1637] V128N,C,Q,S;

[1638] I129N,C,Q,S;

[1639] S130N,Q;

[1640] G131N,C,Q,S,T;

[1641] H133N,C,T,M,W,V,D;

[1642] L134N,C,S,T;

[1643] K136N,C,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1644] W138N,Q,S,T;

[1645] G145N,C,Q,S,T;

[1646] G147N,C,Q,S,T;

[1647] S148C,Q,T;

[1648] T149N,C,Q,S;

[1649] Y150C;

[1650] F153N,C,Q,S,T;

[1651] K154N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1652] W155N,C,Q,S,T;

[1653] H156C,Q,S,T A,I,L,M,F,P,W,V,E;

[1654] W157S,T;

[1655] W165N,C,Q,S,T;

[1656] S168N,C,Q;

[1657] R169AN,C,Q,S A,M,P,W,Y,V,D,E;

[1658] K170N,C,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1659] L171N,C,Q,S,T;

[1660] R173N,C,Q,S A,M,P,W,Y,V,D,E;

[1661] K176N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1662] G179C,Q;

[1663] W182N,C,Q,S,T;

[1664] K180A,I,L,M,F,P,W,Y,V;

[1665] W182N,C,Q,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1666] W184I,L,M,F,P,W,Y,V;

[1667] G191N,Q,S,T;

[1668] L196N,Q,S,T;

[1669] P206N,C,Q,S,T;

[1670] A209S;

[1671] A210N,C,Q;

[1672] R214C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1673] T217N,C,Q,S;

[1674] W218N,C,Q,S,T;

[1675] K234C,S,T A,I,M,F,P,W,Y,V,D;

[1676] H235N,C,Q,S,T A,I,L,M,F,P,W,Y,V;

[1677] R242S,T,I,L,M,F,W,Y,V;

[1678] H247N,C,Q,S,T,I,L,M,F,P,W,V;

[1679] R249N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1680] K251N,C,S,T I,L,M,F,P,W,Y,V,D,E;

[1681] T252N,C,Q,S;

[1682] G253C,Q,S,T;

[1683] K254N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1684] F257N,C,Q,S,T;

[1685] W263N,C,Q,S,T;

[1686] L267N,C,Q,S,T;

[1687] G268N,C,Q,S,T;

[1688] A269N,C,Q,S,T;

[1689] K276N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1690] Q291N;

[1691] H293N,S,T A,I,L,M,P,W,V;

[1692] A294N,Q,S,T;

[1693] G299S,T;

[1694] G300C,T;

[1695] G301N,Q,S,T;

[1696] R305S,T,I,L,M,F,P,W,Y,V;

[1697] K306Q,S,T,I,L,M,F,P,W,Y,V,;

[1698] L308N,C,Q,S,T;

[1699] G310N,C,Q,S,T;

[1700] S314C;

[1701] K315N,C,Q,S,T A,I,L,M,F,P,W,Y,V,E;

[1702] H316N,C,Q,S,T A,I,L,M,F,P,W,V,D,E;

[1703] P317N,C,Q,S,T;

[1704] L318N,C,Q,S;

[1705] K319N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1706] G332N,C,Q,S,T;

[1707] Q333N;

[1708] S334N,C,Q;

[1709] L335C,Q;

[1710] S337N,C,Q;

[1711] T338N,C,Q,S;

[1712] T341C;

[1713] W342S,T;

[1714] P345N,C,Q,T;

[1715] E355N,C,Q,S,T;

[1716] Y358C;

[1717] Y363C;

[1718] K370N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1719] G371N,C,Q,S,T;

[1720] S373N,C,Q,T;

[1721] Q374N;

[1722] R375N,C,Q,S,T A,I,L,M,F,P,W,V,D;

[1723] P378N,C,Q,S,T;

[1724] A379N,C,Q,,T;

[1725] K381N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1726] K389C,S,T,A,M,F,P,W,Y,V,D;

[1727] Q393N;

[1728] Y394C;

[1729] Y396C;

[1730] A398N,C,Q;

[1731] Y402C;

[1732] F403N,Q,S,T;

[1733] H405I,L,M,F,P,W,Y,V;

[1734] H406A,I,M,F,P,Y,V;

[1735] I408N,C,Q,S,T;

[1736] R413N,C,Q,S,T A,I,L,M,F,P,W,Y,V,D,E;

[1737] G415N,C,Q,S,T;

[1738] S417N,Q;

[1739] S418N,Q;

[1740] V419N,C,Q,S,T;

[1741] A420N,C,S,T;

[1742] S422N,Q;

[1743] G431N,Q,S,T;

[1744] P432S,T;

[1745] G433N,Q,S,T;

[1746] G434N,Q,S,T;

[1747] A435Q,T;

[1748] K436N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

[1749] R437N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E;

[1750] Y439C;

[1751] R442N,C,S,T, A,I,L,M,F,P,W,Y,V,D,E;

[1752] Q443N;

[1753] A445N,C,Q,S,T;

[1754] G446N,C,Q,S,T;

[1755] T448N,C,Q,S;

[1756] H450N,C,Q,S,T, A,I,L,M,F,P,W,V,D,E;

[1757] G454N,C,Q,S,T;

[1758] R456C,S,T,A,I,L,M,F,P,W,Y,V,D,E;

[1759] S457N,C,Q;

[1760] P459N,C,Q,S;

[1761] V460N,C,Q,S,T;

[1762] V461N,C,Q,S;

[1763] S464N,C,Q,T;

[1764] W467N,C,Q,S,T;

[1765] G475N,Q,S,T;

[1766] Q482N;

[1767] R483N,C,Q,S,T,A,I,L,M,F,P,W,Y,V,D,E.

[1768] Increased Stability at Low pH and/or at High Temperature

[1769] Substitutions Resulting in a More Hydrophobic Amino Acid Residue

[1770] In an aspect the invention relates to Termamyl-like alpha-amylasevariant with increased stability at acidic pH and/or at high temperaturein comparison to the parent alpha-amylase. Such variants are especiallysuitable for starch liquefaction.

[1771] In the context of the invention the term “acidic pH” means a pHbelow 7.0, especially below the pH range, in which industrial starchliquefaction processes are normally performed, which is between pH 5.5and 6.2.

[1772] In the context of the invention “high temperature” is atemperature in the range from 60-110° C.

[1773] Such variant are variant having a substitution resulting in amore hydrophobic amino acid residues. Providing such variant of theinvention may be prepared by

[1774] 1) Substituting a charged amino acid residue with a hydrophobicamino acid residue;

[1775] 2) Substituting a hydrophilic amino acid residue with ahydrophobic amino acid residue;

[1776] 3) Substituting a hydrophilic amino acid residue with a morehydrophilic amino acid residue;

[1777] 4) Substituting a hydrophilic amino acid residue with a lesshydrophilic amino acid residue.

[1778] Thus, variants of the invention include (using SEQ ID NO: 8 forthe numbering):

[1779] G5A,V,P,M,L,I,Y,F,W;

[1780] T6G,A,V,P,M,L,I,Y,F,W;

[1781] G36A,V,P,M,L,I,Y,F,W;

[1782] I37Y,F,W;

[1783] T38G,A,V,P,M,L,I,Y,F,W;

[1784] A39V,P,M,L,I,Y,F,W;

[1785] I42Y,F,W;

[1786] A45V,P,M,L,I,Y,F,W;

[1787] K47G,A,I,L,M,F,P,W,Y,V;

[1788] D63G,A,V,P,M,L,I,Y,F,W;

[1789] E66G,A,V,P,M,L,I,Y,F,W;

[1790] Q69T,S,C, G,A,V,P,M,L,I,Y,F,W;

[1791] K70G,A,V,P,M,L,I,Y,F,W;

[1792] G71A,V,P,M,L,I,Y,F,W;

[1793] T72G,A,V,P,M,L,I,Y,F,W;

[1794] R74G,A,V,P,M,L,I,Y,F,W;

[1795] T75G,A,V,P,M,L,I,Y,F,W;

[1796] K76G,A,V,P,M,L,I,Y,F,W;

[1797] T79G,A,V,P,M,L,I,Y,F,W:

[1798] E82G,A,V,P,M,L,I,Y,F,W;

[1799] L83I,Y,F,W;

[1800] A86V,P,M,L,I,Y,F,W;

[1801] 187Y,F,W;

[1802] S89G,A,V,P,M,L,I,Y,F,W;

[1803] R93G,A,V,P,M,L,I,Y,F,W;

[1804] T112G,A,V,P,M,L,I,Y,F,W;

[1805] E113G,A,V,P,M,L,I,Y,F,W;

[1806] A117,V,P,M,L,I,Y,F,W;

[1807] V120P,M,L,I,Y,F,W;

[1808] A137V,P,M,L,I,Y,F,W;

[1809] K213G,A,V,P,M,L,I,Y,F,W;

[1810] G216A,V,P,M,L,I,Y,F,W;

[1811] A20V,P,M,L,I,Y,F,W;

[1812] L223I,Y,F,W;

[1813] L225I,Y,F,W;

[1814] D226G,A,V,P,M,L,I,Y,F,W;

[1815] G227G,A,V,P,M,L,I,Y,F,W;

[1816] R229G,A,V,P,M,L,I,Y,F,W;

[1817] D243G,A,V,P,M,L,I,Y,F,W;

[1818] V245P,M,L,I,Y,F,W;

[1819] F279W;

[1820] S282T,G,A,V,P,M,L,I,Y,F,W;

[1821] T311G,A,V,P,M,L,I,Y,F,W;

[1822] V321P,M,L,I,Y,F,W;

[1823] V324P,M,L,I,Y,F,W;

[1824] L352I,Y,F,W;

[1825] T353G,A,V,P,M,L,I,Y,F,W;

[1826] R354G,A,V,P,M,L,I,Y,F,W;

[1827] G357A,V,P,M,L,I,Y,F,W;

[1828] V361P,M,L,I,Y,F,W;

[1829] F362W;

[1830] G364A,V,P,M,L,I,Y,F,W;

[1831] G368A,V,P,M,L,I,Y,F,W;

[1832] A390V,P,M,L,I,Y,F,W;

[1833] A395V,P,M,L,I,Y,F,W;

[1834] G397A,V,P,M,L,I,Y,F,W;

[1835] Q399T,S,C,G,A,V,P,M,L,I,Y,F,W;

[1836] H400G,A,V,P,M,L,I,Y,F,W;

[1837] D401G,A,V,P,M,L,I,Y,F,W;

[1838] A425V,P,M,L,I,Y,F,W;

[1839] D451G,A,V,P,M,L,I,Y,F,W;

[1840] I452Y,F,W;

[1841] T453G,A,V,P,M,L,I,Y,F,W;

[1842] G466G,A,V,P,M,L,I,Y,F,W;

[1843] G468A,V,P,M,L,I,Y,F,W;

[1844] F470W;

[1845] H471G,A,V,P,M,L,I,Y,F,W;

[1846] S478T,G,A,V,P,M,L,I,Y,F,W

[1847] Q9A,C,G,M,P,S,T,W,Y,V;

[1848] F11W;

[1849] E12A,G,I,L,M,F,P,W,Y,V;

[1850] G19A,I,L,M,F,P,W,Y,V;

[1851] H21A,G,I,L,M,F,P,W,Y,V;

[1852] L251,W,Y;

[1853] L321,F,W,Y;

[1854] V40I,L,M,F,P,W,Y;

[1855] Y46W;

[1856] G48M,F,P,W,Y;

[1857] G55AI,L,M,F,P,W,Y,V;

[1858] G57M,P,W;

[1859] A58G,M,W,Y;

[1860] D60AG,I,L,M,F,PW,Y,V;

[1861] Y77W;

[1862] I95F,W,Y;

[1863] V97I,L,M,F,P,W,Y;

[1864] Y98W;

[1865] G99I,L,M,F,P,W,Y,V;

[1866] D100A,G,I,M,F,P,W,Y,V;

[1867] V101I,L,M,P,W,Y;

[1868] V102I,L,M,F,P,W,Y;

[1869] I103W;

[1870] H105A,G,I,L,M,P,Y,V;

[1871] G107M,F,P,W,Y;

[1872] V115I,L,M,F,P,W,Y;

[1873] V118I,L,M,F,P,W,Y;

[1874] I135F,W,Y;

[1875] T139AG,I,L,M,F,P,W,Y,V;

[1876] F141W;

[1877] S151A,G,M,P,T,Y,V;

[1878] H159A,G,I,L,M,F,P,W,V;

[1879] F160W;

[1880] D161A,G,I,L,M,F,P,W,Y,V;

[1881] G162A,I,L,M,F,P,W,Y,V;

[1882] T163A,G,I,L,M,F,P,Y,V;

[1883] D166A,G,I,L,M,F,P,W,Y,V;

[1884] Y175W;

[1885] F177W;

[1886] D183A,G,I,L,M,F,P,W,Y,V;

[1887] V186I,L,M,F,P,W,Y;

[1888] S187A,G,I,L,M,F,P,W,Y,V;

[1889] N192A,G,I,L,M,F,P,W,Y,V;

[1890] A199G,I,L,M,F,P,W,Y,V;

[1891] D200AG,I,L,M,F,P,W,Y,V;

[1892] D202A,G,I,L,M,F,P,W,Y,V;

[1893] V208L,M,F,P,W,Y;

[1894] Y219W;

[1895] F228W;

[1896] L230W,Y;

[1897] V233G,I,M,P,W,Y;

[1898] I236M,P;

[1899] F238W;

[1900] F240W;

[1901] L241P;

[1902] V248A,G,I,L,M,F,P,W,Y;

[1903] T258A,G,I,M,F,P,W,Y,V;

[1904] V259M,P,W,Y;

[1905] A260I,L,M,F,P,W,Y,V;

[1906] Y262W;

[1907] L270I,F,W,Y;

[1908] L274I,M,F,P,W,Y;

[1909] T277A,G,M,P,W,Y,V;

[1910] H281A,G,M,P,W,Y,V;

[1911] V283I,L,M,F,P,W,Y;

[1912] D285A,G,I,M,F,P,W,Y,V;

[1913] V286I,M,F,P,W,Y;

[1914] P287I,L,M,F,W,Y;

[1915] L288I,F,W,Y;

[1916] H289A,G,I,L,M,F,P,W,Y,V;

[1917] F292W;

[1918] A295I,L,M,F,P,W,Y,V;

[1919] S296A,G,I,L,M,F,P,W,T,Y,V;

[1920] L307I,F,W,Y;

[1921] V312I,L,M,F,P,W,Y;

[1922] V313I,L,M,F,P,W,Y;

[1923] S320G,I,L,M,F,P,T,W,Y,V;

[1924] T322G,L,M,F,P,W,Y,V;

[1925] F323W;

[1926] D325AG,I,L,M,F,P,W,Y,V;

[1927] N326A,C,G,M,P,S,T,W;

[1928] H327A,G,I,L,M,P,W,Y,V;

[1929] T329A,G,I,L,M,F,P,W,Y,V;

[1930] P331I,L,M,F,W,Y;

[1931] V339I,L,M,F,P,W,Y;

[1932] K344AG,I,L,M,F,P,W,Y,V;

[1933] L346I,F,W,Y;

[1934] A347I,L,M,F,P,W,Y,V;

[1935] A349I,L,M,F,P,W,Y,V;

[1936] P359I,L,M,F,W,Y;

[1937] Q360I,L,M,F,P,S,T,W,Y,V;

[1938] T369A,G,I,L,M,F,P,W,Y,V;

[1939] I377F,W,Y;

[1940] L380W,Y;

[1941] I387W,Y;

[1942] V409M,P,W,Y;

[1943] G410A,I,L,M,F,P,W,Y,V;

[1944] T412G,I,L,M,F,P,W,Y,V;

[1945] G423A,I,L,M,F,P,W,Y,V;

[1946] L424I,,F,W,Y;

[1947] A426I,L,M,F,P,W,Y,V;

[1948] L427Y;

[1949] I428F,W,Y;

[1950] T429A,G,I,L,M,F,P,W,Y,V;

[1951] D430A,G,I,L,M,F,P,W,Y,V;

[1952] V440I,L,M,F,P,W,Y;

[1953] G441A,I,L,M,F,P,W,Y,V;

[1954] I462F,W,Y;

[1955] V472I,L,M,F,P,W,Y;

[1956] V477I,L,M,F,P,W,Y;

[1957] I479F,W,Y;

[1958] Y480W;

[1959] V481M,P,Y.

[1960] A1I,L,M,F,P,W,Y;

[1961] N2C,Q,I,L,M,F,S,T,W,Y,V;

[1962] L3I,M,F,P,W,Y;

[1963] N4A,C,Q,I,L,M,F,P,S,T,W,Y,V;

[1964] Y14W;

[1965] P16I,L,M,F,W,Y;

[1966] N17A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

[1967] D18A,G,I,L,M,F,P,W,Y,V;

[1968] Q20A,C,I,L,M,F,P,S,T,W,Y,V;

[1969] R23A,G,I,L,M,F,P,W,Y,V;

[1970] R24A,G,I,L,M,F,P,W,Y,V;

[1971] Q26A,C,G,I,L,M,F,P,S,T,W,V;

[1972] E34A,G,I,L,M,F,P,W,Y,V;

[1973] H35A,G,M,F,P,W,Y,V;

[1974] T49A,G,P;

[1975] S50A,G,M,F,P,W;

[1976] Q51A,C,G,I,L,M,F,P,S,T,W,Y,V;

[1977] A52P;

[1978] D53A,G,M,P;

[1979] L61I,M,Y;

[1980] H68A,G,I,L,M,F,P,W,Y,V;

[1981] V73M,P,W,Y;

[1982] Q84A,C,G,I,L,M,F,P,S,T,W,Y,V;

[1983] S85A,G,I,L,M,F,P,T,W,Y,V;

[1984] K88A,G,I,L,M,F,P,W,Y,V;

[1985] H91A,G,I,L,M,F,P,W,Y,V;

[1986] S92A,G,I,L,M,F,P,T,W,Y,V;

[1987] N96A,C,G,I,L,M,F,P,S,T,W,Y,V;

[1988] K106A,G,I,L,M,P,Y,V;

[1989] G108I,L,M,F,P,W,Y,V;

[1990] D114A,G,F,P,W,Y;

[1991] T116A,G,I,L,M,F,P,W,Y,V;

[1992] E119A,G,I,L,M,F,P,W,Y,V;

[1993] D121A,G,I,L,M,F,P,W,Y,V;

[1994] P122I,L,M,F,W,Y;

[1995] A123I,L,M,F,P,W,Y,V;

[1996] D124G,I,L,M,F,P,W,Y,V;

[1997] R125G,I,M,F,W,Y;

[1998] N126Q,G,I,L,M,F,P,S,T,W,Y,V;

[1999] R127A,G,I,L,M,F,P,,W,Y,V;

[2000] V128I,L,M,F,P,W,Y;

[2001] I129F,W,Y;

[2002] S130A,G,I,L,M,F,P,W,Y,V;

[2003] G131A,I,L,M,F,P,W,Y,V;

[2004] E132G,I,L,M,F,W,Y;

[2005] H133M,W,V;

[2006] L134I,F,W,Y;

[2007] K136A,G,I,L,M,F,P,W,Y,V;

[2008] G145A,I,L,M,P,Y,V;

[2009] G147A,I,L,M,P,W,Y,V;

[2010] S148A,G,I,L,M,F,P,T,W,Y,V;

[2011] T149A,G,L,M,F,P,W,Y,V;

[2012] Y150W;

[2013] D152A,G,I,L,M,F,P,W,Y,V;

[2014] F153W;

[2015] K154A,G,I,L,M,F,P,W,Y,V;

[2016] H156A,G,I,L,M,F,P,W,V;

[2017] Y158W;

[2018] D164I,L,M,F,P,W,Y,V;

[2019] E167A,G,I,L,M,F,P,W,Y,V;

[2020] S168A,G,I,L,M,F,T,W,V;

[2021] R169A,G,M,P,W,Y,V;

[2022] K170A,G,I,L,M,F,P,W,Y,V;

[2023] L171W,Y;

[2024] N172A,C,Q,G,I,L,M,F,P,T,W,Y,V;

[2025] R173A,G,M,P,W,Y,V;

[2026] K176A,G,I,L,M,F,P,W,Y,V;

[2027] G179I,L,M,F,P,W,Y,V;

[2028] K180A,G,I,L,M,F,P,W,Y,V;

[2029] E185I,L,M,F,P,W,Y,V;

[2030] N188A,Q,G,L,M,F,W,V;

[2031] E189A,G,I,L,M,F,P,W,Y,V;

[2032] G191A,I,L,M,F,P,W,Y,V;

[2033] Y193W;

[2034] L196I,W;

[2035] Y198W;

[2036] D204L,M,F,P,W,Y,V;

[2037] H205A,G,I,L,M,F,P,W,Y,V;

[2038] P206G,I,L,M,F,W,Y,V;

[2039] A209P,W,Y;

[2040] A210G,I,L,M,F,P,W,Y,V;

[2041] R214A,G,I,L,M,F,P,Y,V;

[2042] T217A,G,I,L,M,F,P,W,Y;

[2043] N221A,C,Q,G,I,L,M,F,P,S,T,W;Y,V;

[2044] E222A,G,I,L,M,F,P,W,Y,V;

[2045] K234A,G,I,M,F,P,W,Y,V;

[2046] H235A,G,I,L,M,F,P,W,Y,V;

[2047] K237A,G,I,L,M,F,W,Y,V;

[2048] S239G,I,L,M,F,P,T,Y,V;

[2049] R242G,I,L,M,F,W,Y,V;

[2050] N246A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

[2051] H247G,I,L,M,F,P,W,V;

[2052] R249A,G,I,L,M,F,P,W,Y,V;

[2053] E250A,I,L,M,P,W,Y,V;

[2054] K251G,I,L,M,F,P,W,Y,V;

[2055] T252A,G,I,L,M,F,P,W,Y,V;

[2056] G253I,L,M,F,P,W,Y;

[2057] K254A,G,I,L,M,F,P,W,Y,V;

[2058] E255A,G,I,L,M,F,W,Y,V;

[2059] F257W;

[2060] E261A,G,I,L,M,F,P,W,Y,V;

[2061] N265C,Q,I,L,M,F,P,W;

[2062] D266A,G,I,L,M,F,P,W,Y,V;

[2063] L267W,Y;

[2064] G268A,I,L,M,F,P,W,Y,V;

[2065] A269I,L,M,F,P,W,Y,V;

[2066] E271A,G,I,L,M,F,P,W,Y,V;

[2067] N272A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

[2068] N275A,C,Q,G,I,L,M,F,P,S,W,Y,V;

[2069] K276A,G,I,L,M,F,P,W,Y,V;

[2070] N278A,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2071] N280A,C,G,I,L,M,F,P,W,Y,V;

[2072] Y290W;

[2073] Q291A,G,I,L,M,F,P,S,T,W,Y,V;

[2074] H293A,G,I,L,M,P,W,V;

[2075] A294I,L,M,F,P,W,Y;

[2076] T297A,G,I,L,M,F,P,W,Y,V;

[2077] Q298G,I,L,M,F,P,S,T,W,Y,V;

[2078] G299A,M,P;

[2079] G300A,I,L,M,F,P,W,Y,V;

[2080] G301I,L,M,F,P,W,Y,V;

[2081] Y302W;

[2082] D303I,L,M,F,P,W,Y,V;

[2083] R305G,I,L,M,F,P,W,Y,V;

[2084] K306G,I,L,M,F,P,W,Y,V;

[2085] L308I,F,W,Y;

[2086] N309Q,G,I,L,M,F,P,S,T,W,Y,V;

[2087] G310A,I,L,M,F,P,W,Y,V;

[2088] S314A,G,I,L,M,F,P,W,Y,V;

[2089] K315A,G,I,L,M,F,P,W,Y,V;

[2090] H316A,G,I,L,M,F,P,W,V;

[2091] P317I,L,M,F,W,Y;

[2092] L318I,W,Y;

[2093] K319A,G,I,L,M,F,P,W,Y,V;

[2094] D328A,G,I,L,M,F,P,W,Y,V;

[2095] G332A,I,L,M,F,P,W,Y,V;

[2096] Q333A,C,G,I,M,F,P,S,T,Y,V;

[2097] S334G,M,F,P,W,Y;

[2098] L335I,F,W,Y;

[2099] E336A,G,I,L,M,F,P,W,Y,V;

[2100] S337A,G,I,L,M,F,P,T,W,Y,V;

[2101] T338A,G,I,L,M,F,P,W,Y,V;

[2102] Q340I,L,M,F,P,S,T,W,Y,V;

[2103] T341A,G,I,L,M,F,W,Y,V;

[2104] P345I,L,M,F,W,Y;

[2105] E355A,G,I,L,M,F,P,W,Y,V;

[2106] Y358W;

[2107] Y363W;

[2108] K370A,G,I,L,M,F,P,W,Y,V;

[2109] G371A,I,L,M,F,P,W,Y,V;

[2110] S373A,G,I,L,M,F,T,W,Y,V;

[2111] Q374A,C,G,I,L,M,F,S,T,W,Y,V;

[2112] R375A,G,I,L,M,F,P,W,V;

[2113] E376A,G,I,L,M,F,P,W,Y,V;

[2114] P378G,I,L,M,F,W,Y,V;

[2115] A379I,L,M,F,P,W,Y,V;

[2116] K381A,G,I,L,M,F,P,W,Y,V;

[2117] K389A,G,M,F,P,W,Y,V;

[2118] Q393A,C,G,I,L,M,F,P,S,T,W,Y,V;

[2119] Y394W;

[2120] Y396W;

[2121] A398I,L,M,F,W,Y,V;

[2122] Y402W;

[2123] F403W;

[2124] D404I,L,M,F,P,W,Y,V;

[2125] H405G,I,L,M,F,P,W,Y,V;

[2126] H406A,G,I,M,F,P,Y,V;

[2127] D407A,G,I,L,M,F,P,W,Y,V;

[2128] I408W,Y;

[2129] R413A,G,I,L,M,F,P,W,Y,V;

[2130] E414A,G,I,L,M,F,P,W,Y,V;

[2131] G415A,I,L,M,F,P,W,Y,V;

[2132] D416A,G,I,L,M,F,P,W,Y,V;

[2133] S417A,G,I,L,M,F,P,W,Y,V;

[2134] S418A,G,I,L,M,F,P,T,W,Y,V;

[2135] V419I,L,M,F,P,W,Y;

[2136] A420I,L,M,F,W,Y,V;

[2137] N421A,C,Q,I,L,M,F,P,S,T,W,Y,V;

[2138] S422A,G,I,L,M,F,P,T,W,Y,V;

[2139] G431A,I,L,M,F,P,W,Y,V;

[2140] P432I,L,M,F,W,Y;

[2141] G433A,I,L,M,F,P,W,Y,V;

[2142] G434A,I,L,M,F,P,W,Y,V;

[2143] A435I,L,M,F,P,W,Y,V;

[2144] K436A,G,I,L,M,F,P,W,Y,V;

[2145] R437A,G,I,L,M,F,P,W,Y,V;

[2146] Y439W;

[2147] R442A,G,I,L,M,F,P,W,Y,V;

[2148] Q443A,C,G,I,L,M,F,P,S,T,W,Y,V;

[2149] N444A,C,G,I,L,M,F,P,S,T,W,Y,V;

[2150] A445I,L,M,F,P,W,Y,V;

[2151] G446A,I,L,M,F,P,W,Y,V;

[2152] E447A,G,I,L,M,F,P,W,Y,V;

[2153] T448A,G,I,L,M,F,P,W,Y,V;

[2154] H450A,G,I,L,M,F,P,W,V;

[2155] G454A,I,L,M,F,P,W,Y,V;

[2156] N455A,C,Q,G,I,L,M,F,P,S,T,W,Y,V;

[2157] R456A,I,L,M,F,P,W,Y,V;

[2158] S457A,G,I,L,M,F,W,Y,V;

[2159] E458A.G,I,L,M,F,P,W,Y,V;

[2160] P459I,L,M,F,W,Y;

[2161] V460I,L,M,F,P,W,Y;

[2162] V461I,L,M,F,P,W,Y;

[2163] N463A,C,Q,I,L,M,F,P,S,T,W,Y,V;

[2164] S464A,G,I,L,M,F,P,T,W,Y,V;

[2165] E465A,G,I,L,M,F,P,W,Y,V;

[2166] E469A,G,I,L,M,F,P,W,Y,V;

[2167] N473Q,G,I,L,M,F,P,S,T,W,Y,V;

[2168] G474A,I,L,M,F,P,W,Y,V;

[2169] G475A,I,L,M,F,P,W,Y,V;

[2170] S476G,I,L,M,F,P,T,W,Y,V;

[2171] Q482A,C,G,I,L,M,F,S,T,W,Y,V;

[2172] R483A,G,I,L,M,F,P,W,Y,V.

[2173] General Mutations in Variants of the Invention

[2174] A variant of the invention may in one embodiment comprise one ormore modifications in addition to those outlined above. Thus, it may beadvantageous that one or more Proline (Pro) residues present in the partof the alpha-amylase variant which is modified is/are replaced with anon-Proline residue which may be any of the possible, naturallyoccurring non-Proline residues, and which preferably is an Alanine,Glycine, Serine, Threonine, Valine or Leucine.

[2175] Analogously, in one embodiment one or more Cysteine residuespresent in the parent alpha-amylase may be replaced with a non-Cysteineresidue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.

[2176] Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino add fragment 185-209of SEQ ID NO: 10 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like alpha-amylase, of oneor more of the Lys residues present in an amino acid fragmentcorresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by anArg.

[2177] It is to be understood that the present invention encompassesvariants incorporating two or more of the above outlined modifications.

[2178] Furthermore, it may be advantageous to introduce mutations in oneor more of the following positions (using SEQ ID NO: 8 (Termamyl) forthe numbering):

[2179] M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405,T412, in particular the following single, double or triple or multimutations:

[2180] M15X, in particular M15T,L;

[2181] V128X, in particular V128E;

[2182] H133X, in particular H133Y;

[2183] N188X, in particular N188S,T,P;

[2184] M197X, in particular M197T,L;

[2185] A209X, in particular A209V;

[2186] M197T/W138F; M197T/138Y; M15T/H133Y/N188S;

[2187] M15N128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T149I;

[2188] G475R, H133Y/S187D; H133Y/A209V.

[2189] Methods for Preparing Alpha-Amylase Variants of the Invention

[2190] Several methods for introducing mutations into genes are known inthe art. After a brief description of cloning of alpha-amylase-encodingDNA sequences, methods for generating mutations at specific sites withinthe alpha-amylase-encoding sequence will be discribed.

[2191] Cloning a DNA Sequence Encoding an Alpha-Amylase

[2192] The DNA sequence encoding a parent alpha-amylase may be isolatedfrom any cell or microorganism producing the alpha-amylase in question,using various methods well known in the art. First, a genomic DNA and/orcDNA library should be constructed using chromosomal DNA or messengerRNA from the organism that produces the alpha-amylase to be studied.Then, if the amino acid sequence of the alpha-amylase is known,homologous, labeled oligonucleotide probes may be synthesized and usedto identify alpha-amylase-encoding clones from a genomic libraryprepared from the organism in question. Alternatively, a labeledoligonucleotide probe containing sequences homologous to a knownalpha-amylase gene could be used as a probe to identifyalpha-amylase-encoding clones, using hybridization and washingconditions of lower stringency.

[2193] Yet another method for identifying alpha-amylase-encoding cloneswould involve inserting fragments of genomic DNA into an expressionvector, such as a plasmid, transforming alpha-amylase-negative bacteriawith the resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for alpha-amylase, therebyallowing clones expressing the alpha-amylase to be identified.

[2194] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described byMatthes et al., The EMBO J. 3, 1984, pp. 801-805. In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

[2195] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligabng fragments of synthetic, genomic or cDNAorigin (as appropriate, the fragments corresponding to various parts ofthe entire DNA sequence), in accordance with standard techniques. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al., Science 239, 1988, pp. 487-491.

[2196] Site-Directed Mutagenesis

[2197] Once an alpha-amylase-encoding DNA sequence has been isolated,and desirable sites for mutation identified, mutations may be introducedusing synthetic oligonucleotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-stranded DNAThe remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. (1984). U.S. Pat.No. 4,760,025 disclose the introduction of oligonucleotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

[2198] Another method for introducing mutations intoalpha-amylase-encoding DNA sequences is described in Nelson and Long(1989). It involves the 3-step generation of a PCR fragment containingthe desired mutation introduced by using a chemically synthesized DNAstrand as one of the primers in the PCR reactions. From thePCR-generated fragment, a DNA fragment carrying the mutation may beisolated by cleavage with restriction endonucleases and reinserted intoan expression plasmid.

[2199] Alternative methods for providing variants of the inventioninclude gene shuffling, e.g., as described in WO 95/22625 (from AffymaxTechnologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S), or othercorresponding techniques resulting in a hybrid enzyme comprising themutation(s), e.g., substitution(s) and/or deletion(s), in question.

[2200] Expression of Alpha-Amylase Variants

[2201] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in enzyme form, using an expression vectorwhich typically includes control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes.

[2202] The recombinant expression vector carrying the DNA sequenceencoding an alpha-amylase variant of the invention may be any vector,which may conveniently be subjected to recombinant DNA procedures, andthe choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

[2203] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E.coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

[2204] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding thealpha-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

[2205] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and pIJ702.

[2206] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

[2207] While intracellular expression may be advantageous in somerespects, e.g., when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular. In general,the Bacillus alpha-amylases mentioned herein comprise a preregionpermitting secretion of the expressed protease into the culture medium.If desirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

[2208] The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

[2209] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of analpha-amylase variant of the invention. The cell may be transformed withthe DNA construct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

[2210] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

[2211] Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such asE.coli. The transformation of the bacteria may, for instance, beeffected by protoplast transformation or by using competent cells in amanner known per se.

[2212] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

[2213] In a yet further aspect, the present invention relates to amethod of producing an alpha-amylase variant of the invention, whichmethod comprises cultivating a host cell as described above underconditions conducive to the production of the variant and recovering thevariant from the cells and/or culture medium.

[2214] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the alpha-amylase variant of the invention. Suitable mediaare available from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

[2215] The alpha-amylase variant secreted from the host cells mayconveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulphate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

[2216] Industrial Applications

[2217] The alpha-amylase variants of this invention possess valuableproperties allowing for a variety of industrial applications. Inparticular, enzyme variants of the invention are applicable as acomponent in washing, dishwashing, and hard surface cleaning detergentcompositions.

[2218] Variant of the invention with altered properties may be used forstarch processes, in particular starch conversion, especiallyliquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP patentapplication nos. 252 730 and 63 909, WO 99/19467, and WO 96/28567 allreferences hereby incorporated by reference). Also contemplated arecompositions for starch conversion purposes, which may beside thevariant of the invention also comprise a glucoamylase, pullulanase, andother alpha-amylases.

[2219] Further, variants of the invention are also particularly usefulin the production of sweeteners and ethanol (see, e.g., U.S. Pat. No.5,231,017 hereby incorporated by reference), such as fuel, drinking andindustrial ethanol, from starch or whole grains.

[2220] Variants of the invention may also be useful for desizing oftextiles, fabrics and garments (see, e.g., WO 95/21247, U.S. Pat. No.4,643,736, EP 119,920 hereby in corporate by reference), beer making orbrewing, in pulp and paper production.

[2221] Starch Conversion

[2222] Conventional starch-conversion processes, such as liquefactionand saccharification processes are described, e.g., in U.S. Pat. No.3,912,590 and EP patent publications Nos. 252,730 and 63,909, herebyincorporated by reference.

[2223] In an embodiment the starch conversion process degrading starchto lower molecular weight carbohydrate components such as sugars or fatreplacers includes a debranching step.

[2224] Starch to Sugar Conversion

[2225] In the case of converting starch into a sugar the starch isdepolymerized. A such depolymerization process consists of aPre-treatment step and two or three consecutive process steps, viz aliquefaction process, a saccharification process and dependent on thedesired end product optionally an isomerization process.

[2226] Pre-Treatment of Native Starch

[2227] Native starch consists of microscopic granules, which areinsoluble in water at room temperature. When an aqueous starch slurry isheated, the granules swell and eventually burst, dispersing the starchmolecules into the solution. During this “gelatinization” process thereis a dramatic increase in viscosity. As the solids level is 30-40% in atypically industrial process, the starch has to be thinned or“liquefied” so that it can be handled. This reduction in viscosity istoday mostly obtained by enzymatic degradation.

[2228] Liquefaction

[2229] During the liquefaction step, the long chained starch is degradedinto branched and linear shorter units (maltodextrins) by analpha-amylase. The liquefaction process is carried out at 105-110° C.for 5 to 10 minutes followed by 1-2 hours at 95° C. The pH lies between5.5 and 6.2. In order to ensure optimal enzyme stability under theseconditions, 1 mM of calcium is added (40 ppm free calcium ions). Afterthis treatment the liquefied starch will have a “dextrose equivalent”(DE) of 10-15.

[2230] Saccharification

[2231] After the liquefaction process the maltodextrins are convertedinto dextrose by addition of a glucoamylase (e.g., AMG) and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase (e.g., Promozyme™) (U.S. Pat. No. 4,560,651). Before thisstep the pH is reduced to a value below 4.5, maintaining the hightemperature (above 95° C.) to inactivate the liquefying alpha-amylase toreduce the formation of short oligosaccharide called “panose precursors”which cannot be hydrolyzed properly by the debranching enzyme.

[2232] The temperature is lowered to 60° C., and glucoamylase anddebranching enzyme are added. The saccharification process proceeds for24-72 hours.

[2233] Normally, when denaturing the α-amylase after the liquefactionstep about 0.2-0.5% of the saccharification product is the branchedtrisaccharide 6²-alpha-glucosyl maltose (panose) which cannot bedegraded by a pullulanase. If active amylase from the liquefaction stepis present during saccharification (i.e., no denaturing), this level canbe as high as 1-2%, which is highly undesirable as it lowers thesaccharification yield significantly.

[2234] Isomerization

[2235] When the desired final sugar product is, e.g., high fructosesyrup the dextrose syrup may be converted into fructose. After thesaccharification process the pH is increased to a value in the range of6-8, preferably pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immmobilized glucoseisomerase (such as Sweetzyme™ IT).

[2236] Ethanol Production

[2237] In general alcohol production (ethanol) from whole grain can beseparated into 4 main steps

[2238] Milling

[2239] Liquefaction

[2240] Saccharification

[2241] Fermentation

[2242] Milling

[2243] The grain is milled in order to open up the structure andallowing for further processing. Two processes are used wet or drymilling. In dry milling the whole kernel is milled and used in theremaining part of the process. Wet milling gives a very good separationof germ and meal (starch granules and protein) and is with a fewexceptions applied at locations where there is a parallel production ofsyrups.

[2244] Liquefaction

[2245] In the liquefaction process the starch granules are solubilizedby hydrolysis to maltodextrins mostly of a DP higher than 4. Thehydrolysis may be carried out by acid treatment or enzymatically byalpha-amylase. Acid hydrolysis is used on a limited basis. The rawmaterial can be milled whole grain or a side stream from starchprocessing.

[2246] Enzymatic liquefaction is typically carried out as a three-stephot slurry process. The slurry is heated to between 60-95° C.,preferably 80-85° C., and the enzyme(s) is (are) added. Then the slurryis jet-cooked at between 95-140° C, preferably 105-125° C., cooled to60-95° C. and more enzyme(s) is (are) added to obtain the finalhydrolysis. The liquefaction process is carried out at pH 4.5-6.5,typically at a pH between 5 and 6. Milled and liquefied grain is alsoknown as mash.

[2247] Saccharification

[2248] To produce low molecular sugars DP₁₋₃ that can be metabolized byyeast, the maltodextrin from the liquefaction must be furtherhydrolyzed. The hydrolysis is typically done enzymatically byglucoamylases, alternatively alphaglucosidases or acid alpha-amylasescan be used. A full saccharification step may last up to 72 hours,however, it is common only to do a pre-saccharification of typically40-90 minutes and then complete saccharification during fermentation(SSF). Saccharification is typically carried out at temperatures from30-65□C., typically around 60□C., and at pH 4.5.

[2249] Fermentation

[2250] Yeast typically from Saccharomyces spp. is added to the mash andthe fermentation is ongoing for 24-96 hours, such as typically 35-60hours. The temperature is between 26-34° C., typically at about 32° C.,and the pH is from pH 3-6, preferably around pH 4-5.

[2251] Note that the most widely used process is a simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzyme isadded together. When doing SSF it is common to introduce apre-saccharification step at a temperature above 50° C., just prior tothe fermentation.

[2252] Distillation

[2253] Following the fermentation the mash is distilled to extract theethanol.

[2254] The ethanol obtained according to the process of the inventionmay be used as, e.g., fuel ethanol; drinking ethanol, i.e., potableneutral spirits; or industrial ethanol.

[2255] By-Products

[2256] Left over from the fermentation is the grain, which is typicallyused for animal feed either in liquid form or dried.

[2257] Further details on how to carry out liquefaction,saccharification, fermentation, distillation, and recovering of ethanolare well known to the skilled person.

[2258] According to the process of the invention the saccharificationand fermentation may be carried out simultaneously or separately.

[2259] Pulp and Paper Production

[2260] The alkaline alpha-amylase of the invention may also be used inthe production of lignocellulosic materials, such as pulp, paper andcardboard, from starch reinforced waste paper and cardboard, especiallywhere repulping occurs at pH above 7 and where amylases facilitate thedisintegration of the waste material through degradation of thereinforcing starch. The alpha-amylase of the invention is especiallyuseful in a process for producing a papermaking pulp from starch-coatedprinted-paper. The process may be performed as described in WO 95/14807,comprising the following steps:

[2261] a) disintegrating the paper to produce a pulp,

[2262] b) treating with a starch-degrading enzyme before, during orafter step a), and

[2263] c) separating ink particles from the pulp after steps a) and b).

[2264] The alpha-amylases of the invention may also be very useful inmodifying starch where enzymatically modified starch is used inpapermaking together with alkaline fillers such as calcium carbonate,kaolin and clays. With the alkaline alpha-amylases of the invention itbecomes possible to modify the starch in the presence of the filler thusallowing for a simpler integrated process.

[2265] Desizing of Textiles, Fabrics and Garments

[2266] An alpha-amylase of the invention may also be very useful intextile, fabric or garment desizing. In the textile processing industry,alpha-amylases are traditionally used as auxiliaries in the desizingprocess to facilitate the removal of starch-containing size, which hasserved as a protective coating on weft yams during weaving. Completeremoval of the size coating after weaving is important to ensure optimumresults in the subsequent processes, in which the fabric is scoured,bleached and dyed. Enzymatic starch breakdown is preferred because itdoes not involve any harmful effect on the fiber material. In order toreduce processing cost and increase mill throughput, the desizingprocessing is sometimes combined with the scouring and bleaching steps.In such cases, non-enzymatic auxiliaries such as alkali or oxidationagents are typically used to break down the starch, because traditionalalpha-amylases are not very compatible with high pH levels and bleachingagents. The non-enzymatic breakdown of the starch size does lead to somefiber damage because of the rather aggressive chemicals used.Accordingly, it would be desirable to use the alpha-amylases of theinvention as they have an improved performance in alkaline solutions.The alpha-amylases may be used alone or in combination with a cellulasewhen desizing cellulose-containing fabric or textile.

[2267] Desizing and bleaching processes are well known in the art. Forinstance, such processes are described in WO 95/21247, U.S. Pat. No.4,643,736, EP 119,920 hereby in corporate by reference.

[2268] Commercially available products for desizing include AQUAZYME®and AQUAZYME® ULTRA from Novozymes A/S.

[2269] Beer Making

[2270] The alpha-amylases of the invention may also be very useful in abeer-making process; the alpha-amylases will typically be added duringthe mashing process.

[2271] Detergent Compositions

[2272] The alpha-amylase of the invention may be added to and thusbecome a component of a detergent composition.

[2273] The detergent composition of the invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pretreatment of stainedfabrics and a rinse added fabric softener composition or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

[2274] In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a peroxidase, another amylolytic enzyme, e.g.,another alpha-amylase, glucoamylase, maltogenic amylase, CGTase and/or acellulase mannanase (such as MANNAWAY™ from Novozymes, Denmark)),pectinase, pectine lyase, cutinase, and/or laccase.

[2275] In general the properties of the chosen enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

[2276] Proteases: Suitable proteases include those of animal, vegetableor microbial origin. Microbial origin is preferred. Chemically modifiedor protein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likepro-teases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

[2277] Examples of useful proteases are the variants described in WO92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially thevariants with substitutions in one or more of the following positions:27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218,222, 224, 235 and 274.

[2278] Preferred commercially available protease enzymes includeALCALASE®, SAVINASE®, PRIMASE®, DURALASE®, ESPERASE®, and KANNASE® (fromNovozymes A/S), MAXATASE®, MAXACAL, MAXAPEM®, PROPERASE®, PURAFECT®,PURAFECT OXP®, FN2®, FN3®, FN4® (Genencor International Inc.).

[2279] Lipases: Suitable lipases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Examples of useful lipases include lipases from Humicola (synonymThermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described inEP 258 068 and EP 305 216 or from H. insolens as described in WO96/13580, a Pseudomonas lipase, e.g., from P. alcaligenes or P.pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720and WO 96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase,e.g., from B. subtilis (Dartois et al. (1993), Biochemica et BiophysicaActa, 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus(WO 91/16422). Other examples are lipase variants such as thosedescribed in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO95/22615, WO 97/04079 and WO 97/07202.

[2280] Preferred commercially available lipase enzymes include LIPOLASE™and LIPOLASE ULTRA™ (Novozymes A/S).

[2281] Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, alpha-amylasesobtained from Bacillus, e.g., a special strain of B. licheniformis,described in more detail in GB 1,296,839. Examples of usefulalpha-amylases are the variants described in WO 94/02597, WO 94/18314,WO 96/23873, and WO 97/43424, especially the variants with substitutionsin one or more of the following positions: 15, 23, 105, 106, 124, 128,133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305,391, 408, and 444.

[2282] Commercially available alpha-amylases are DURAMYL™, LlQUEZYME™TERMAMY™, NATALASE™, FUNGAMYL™ and BAN™ (Novozymes A/S), RAPIDASE™ andPURASTAR™ (from Genencor International Inc.).

[2283] Cellulases: Suitable cellulases include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Suitable cellulases include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g.,the fungal cellulases produced from Humicola insolens, Myceliophthorathermophila and Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307,U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No.5,776,757 and WO 89/09259.

[2284] Especially suitable cellulases are the alkaline or neutralcellulases having colour care benefits. Examples of such cellulases arecellulases described in EP 0 49.5 257, EP 0 531 372, WO 96/11262, WO96/29397, WO 98/08940. Other examples are cellulase variants such asthose described in WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046,U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO98/12307 and PCT/DK98/00299.

[2285] Commercially available cellulases include CELLUZYME®, andCAREZYME® (Novozymes A/S), CLAZINASE®, and PURADAX HA® (GenencorInternational Inc.), and KAC-500(B)® (Kao Corporation).

[2286] Peroxidases/Oxidases: Suitable peroxidases/oxidases include thoseof plant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

[2287] Commercially available peroxidases include GUARDZYME® (NovozymesA/S).

[2288] The detergent enzyme(s) may be included in a detergentcomposition by adding separate additives containing one or more enzymes,or by adding a combined additive comprising all of these enzymes. Adetergent additive of the invention, i.e., a separate additive or acombined additive, can be formulated, e.g., granulate, a liquid, aslurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

[2289] Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated bymethods known in the art. Examples of waxy coating materials arepoly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molarweights of 1000 to 20000; ethoxylated nonyl-phenols having from 16 to 50ethylene oxide units; ethoxylated fatty alcohols in which the alcoholcontains from 12 to 20 carbon atoms and in which there are 15 to 80ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

[2290] The detergent composition of the invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

[2291] The detergent composition comprises one or more surfactants,which may be non-ionic including semi-polar and/or anionic and/orcationic and/or zwitterionic. The surfactants are typically present at alevel of from 0.1% to 60% by weight.

[2292] When included therein the detergent will usually contain fromabout 1% to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

[2293] When included therein the detergent will usually contain fromabout 0.2% to about 40% of a non-ionic surfactant such as alcoholethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside,alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide,fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, orN-acyl N-alkyl derivatives of glucosamine (“glucamides”).

[2294] The detergent may contain 0-65% of a detergent builder orcomplexing agent such as zeolite, diphosphate, tripho-sphate,phosphonate, carbonate, citrate, nitrilotriacetic acid,ethylenediaminetetraacetic acid, diethylenetri-aminepen-taacetic acid,alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates(e.g. SKS-6 from Hoechst).

[2295] The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid co-polymers.

[2296] The detergent may contain a bleaching system, which may comprisea H₂O₂ source such as perborate or percarbonate which may be combinedwith a peracid-forming bleach activator such astetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate.Alternatively, the bleaching system may comprise peroxyacids of, e.g.,the amide, imide, or sulfone type.

[2297] The enzyme(s) of the detergent composition of the inven-tion maybe stabilized using conventional stabilizing agents, e.g., a polyol suchas propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the com-position may be formulated as described in, e.g., WO92/19709 and WO 92/19708.

[2298] The detergent may also contain other conventional detergentingredients such as e.g. fabric conditioners including clays, foamboosters, suds suppressors, anti-corrosion agents, soil-suspendingagents, anti-soil re-deposition agents, dyes, bactericides, opticalbrighteners, hydrotropes, tarnish inhibitors, or perfumes.

[2299] It is at present contemplated that in the detergent compositionsany enzyme, in particular the enzyme of the invention, may be added inan amount corresponding to 0.01-100 mg of enzyme protein per liter ofwash liquor, preferably 0.055 mg of enzyme protein per liter of washliquor, in particular 0.1-1 mg of enzyme protein per liter of washliquor.

[2300] The enzyme of the invention may additionally be incorporated inthe detergent formulations disclosed in WO 97/07202, which is herebyincorporated as reference.

[2301] Dishwash Deterget Compositions

[2302] The enzyme of the invention mat also be used in dish washdetergent compositions, including the following: 1) POWDER AUTOMATICDISHWASHING COMPOSITION Nonionic surfactant 0.4-2.5% Sodium metasilicate0-20% Sodium disilicate 3-20% Sodium triphosphate 20-40% Sodiumcarbonate 0-20% Sodium perborate 2-9% Tetraacetyl ethylene diamine(TAED) 1-4% Sodium sulphate 5-33% Enzymes 0.0001-0.1%

[2303] 2) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant1-2% (e.g. alcohol ethoxylate) Sodium disilicate 2-30% Sodium carbonate10-50% Sodium phosphonate 0-5% Trisodium citrate dihydrate 9-30%Nitrilotrisodium acetate (NTA) 0-20% Sodium perborate monohydrate 5-10%Tetraacetyl ethylene diamine (TAED) 1-2% Polyacrylate polymer 6-25%(e.g. maleic acid/acrylic acid copolymer) Enzymes 0.0001-0.1% Perfume0.1-0.5% Water 5-10

[2304] 3) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant0.5-2.0% Sodium disilicate 25-40% Sodium citrate 30-55% Sodium carbonate0-29% Sodium bicarbonate 0-20% Sodium perborate monohydrate 0-15%Tetraacetyl ethylene diamine (TAED) 0-6% Maleic acid/acrylic 0-5% acidcopolymer Clay 1-3% Polyamino acids 0-20% Sodium polyacrylate 0-8%Enzymes 0.0001-0.1%

[2305] 4) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant1-2% Zeolite MAP 15-42% Sodium disilicate 30-34% Sodium citrate 0-12%Sodium carbonate 0-20% Sodium perborate monohydrate 7-15% Tetraacetylethylene 0-3% diamine (TAED) Polymer 0-4% Maleic acid/acrylic acidcopolymer 0-5% Organic phosphonate 0-4% Clay 1-2% Enzymes 0.0001-0.1%Sodium sulphate Balance

[2306] 5) POWDER AUTOMATIC DISHWASHING COMPOSITION Nonionic surfactant1-7% Sodium disilicate 18-30% Trisodium citrate 10-24% Sodium carbonate12-20% Monopersulphate (2 KHSO₅.KHSO₄.K₂SO₄) 15-21% Bleach stabilizer0.1-2% Maleic acid/acrylic acid copolymer 0-6% Diethylene triaminepentaacetate, 0-2.5% pentasodium salt Enzymes 0.0001-0.1% Sodiumsulphate, water Balance

[2307] 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANINGSURFACTANT SYSTEM Nonionic surfactant 0-1.5% Octadecyl dimethylamineN-oxide dihydrate 0-5% 80:20 wt. C18/C16 blend of octadecyldimethylamine 0-4% N-oxide dihydrate and hexadecyldimethyl amine N-oxide dihydrate 70:30 wt. C18/C16 blend of octadecyl bis 0-5%(hydroxyethyl)amine N-oxide anhydrous and hexadecyl bis(hydroxyethyl)amine N-oxide anhydrous C₁₃-C₁₅ alkyl ethoxysulfate withan average degree of 0-10% ethoxylation of 3 C₁₂-C₁₅ alkyl ethoxysulfatewith an average degree of 0-5% ethoxylation of 3 C₁₃-C₁₅ ethoxylatedalcohol with an average degree of 0-5% ethoxylation of 12 A blend ofC₁₂-C₁₅ ethoxylated alcohols with an 0-6.5% average degree ofethoxylation of 9 A blend of C₁₃-C₁₅ ethoxylated alcohols with an 0-4%average degree of ethoxylation of 30 Sodium disilicate 0-33% Sodiumtripolyphosphate 0-46% Sodium citrate 0-28% Citric acid 0-29% Sodiumcarbonate 0-20% Sodium perborate monohydrate 0-11.5% Tetraacetylethylene diamine (TAED) 0-4% Maleic acid/acrylic acid copolymer 0-7.5%Sodium sulphate 0-12.5% Enzymes 0.0001-0.1%

[2308] 7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION Liquidnonionic surfactant (e.g. alcohol ethoxylates) 2.0-10.0% Alkali metalsilicate 3.0-15.0% Alkali metal phosphate 20.0-40.0% Liquid carrierselected from higher 25.0-45.0% glycols, polyglycols, polyoxides,glycolethers Stabilizer (e.g. a partial ester of phosphoric acid and a0.5-7.0% C₁₆-C₁₈ alkanol) Foam suppressor (e.g. silicone) 0-1.5% Enzymes0.0001-0.1%

[2309] 8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION Liquid nonionicsurfactant (e.g. alcohol ethoxylates) 2.0-10.0% Sodium silicate3.0-15.0% Alkali metal carbonate 7.0-20.0% Sodium citrate 0.0-1.5%Stabilizing system (e.g. mixtures of finely divided 0.5-7.0% siliconeand low molecular weight dialkyl polyglycol ethers) Low molecule weightpolyacrylate polymer 5.0-15.0% Clay gel thickener (e.g. bentonite)0.0-10.0% Hydroxypropyl cellulose polymer 0.0-0.6% Enzymes 0.0001-0.1%Liquid carrier selected from higher lycols, polyglycols, Balancepolyoxides and glycol ethers

[2310] 9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION C₁₂-C₁₄fatty acid 0-0.5% Block co-polymer surfactant 1.5-15.0% Sodium citrate0-12% Sodium tripolyphosphate 0-15% Sodium carbonate 0-8% Aluminiumtristearate 0-0.1% Sodium cumene sulphonate 0-1.7% Polyacrylatethickener 1.32-2.5% Sodium polyacrylate 2.4-6.0% Boric acid 0-4.0%Sodium formate 0-0.45% Calcium formate 0-0.2% Sodium n-decydiphenyloxide disulphonate 0-4.0% Monoethanol amine (MEA) 0-1.86% Sodiumhydroxide (50%) 1.9-9.3% 1,2-Propanediol 0-9.4% Enzymes 0.0001-0.1% Sudssuppressor, dye, perfumes, water Balance

[2311] 10) LIQUID AUTOMATIC DISHWASHING COMPOSITION Alcohol ethoxylate0-20% Fatty acid ester sulphonate 0-30% Sodium dodecyl sulphate 0-20%Alkyl polyglycoside 0-21% Oleic acid 0-10% Sodium disilicate monohydrate18-33% Sodium citrate dihydrate 18-33% Sodium stearate 0-2.5% Sodiumperborate monohydrate 0-13% Tetraacetyl ethylene diamine (TAED) 0-8%Maleic acid/acrylic acid copolymer 4-8% Enzymes 0.0001-0.1%

[2312] 11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTEDBLEACH PARTICLES Sodium silicate  5-10% Tetrapotassium pyrophosphate15-25% Sodium triphosphate 0-2% Potassium carbonate 4-8% Protectedbleach particles, e.g. chlorine  5-10% Polymeric thickener 0.7-1.5%Potassium hydroxide 0-2% Enzymes 0.0001-0.1%   Water Balance

[2313] 11) Automatic dishwashing compositions as described in 1), 2),3), 4), 6) and 10), wherein perborate is replaced by percarbonate.

[2314] 12) Automatic dishwashing compositions as described in 1)-6)which additionally contain a manganese catalyst. The manganese catalystmay, e.g., be one of the compounds described in “Effident manganesecatalysts for low-temperature bleaching”, Nature 369, 1994, pp. 637-639.

[2315] Uses

[2316] The present invention is also directed to methods for using analpha-amylase variant of the invention in detergents, in particularlaundry detergent compositions and dishwashing detergent compositions,hard surface cleaning compositions, and in composition for desizing oftextiles, fabrics or garments, for production of pulp and paper, beermaking, ethanol production, and starch conversion processes as describedabove.

[2317] Compositions

[2318] The invention also related to composition comprising a variant ofthe invention, and in a preferred embodiment also a B.stearothermophilus alpha-amylase (BSG), in particular a variant thereof.

[2319] In another embodient the composition comprises beside a variantof the invention a glucoamylase, in particular a glucoamylaseoriginating from Aspergillus niger (e.g., the G1 or G2 A. niger AMGdisclosed in Boel et al. (1984), “Glucoamylases G1 and G2 fromAspergillus niger are synthesized from two different but closely relatedmRNAs”, EMBO J. 3 (5), p. 1097-1102, or a variant therefore, inparticular a variant disclosed in WO 00/04136 or WO 01/04273 or theTalaromyces emersonii AMG disclosed in WO 99/28448.

[2320] In an embodiment the composition of the invention also comprisesa pullulanase, in particular a Bacillus pullulanase.

[2321] Materials and Methods

[2322] Materials

[2323]Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 andalso available from Novozymes A/S, Denmark.

[2324] M560: SEQ ID NO: 12; disclosed in WO 00/60060; deposited on Jan.25, 1999 at DSMZ and assigned the DSMZ no. 12649.

[2325] LB medium (In 1 liter H₂O: 10 g bacto-tryptone, 5 g bacto-yeastextract, 10 g NaCl, pH adjusted to 7.0 w. NaOH, autoclaved).

[2326] TY agar plates (In 1 liter H₂O: 16 g bacto-tryptone, 10 gbacto-yeast extract, 5 g NaCl, pH adjusted to 7.0 w. NaOH, and 15 gbacto-agar is added prior to autoclaving).

[2327] 10% Lugol solution (Iodine/Potassium iodine solution; made by10-fold dil. in H₂O of stock: Sigma Cat. no. L 6146).

[2328]Bacillus subtilis SHA273: see WO 95/10603

[2329] Detergents

[2330] Model detergent: A/P (Asia/Pacific) Model Detergent has thefollowing composition: 20% STPP (sodium tripolyphosphate), 25% Na₂SO₄,15% Na₂CO₃, 20% LAS (linear alkylbenzene sulfonate, Nansa 80S), 5%C12-C15 alcohol ethoxylate (Dobanol 25-7), 5% Na₂Si2O₅, 0.3% NaCl.

[2331] Omo Multi Acao (Brazil),

[2332] Omo concentrated powder (EU) (Unilever)

[2333] Ariel Futur liquid (EU) (Procter and Gamble)

[2334] Ariel Essential (EU) (Procter and Gamble)

[2335] Plasmids

[2336] pDN1528 contains the complete gene encoding Termamyl, amyL, theexpression of which is directed by its own promoter. Further, theplasmid contains the origin of replication, ori, from plasmid pUB110 andthe cat gene from plasmid pC194 conferring resistance towardschloramphenicol. pDN1528 is shown in FIG. 9 of WO 96/23874.

[2337] Methods

[2338] Filter Screening Assays

[2339] The below assays can be used to screening of Termamyl-likealpha-amylase variants having altered stability at high or low pH and/orunder Ca²⁺ depleted conditions compared to the parent enzyme andTermamyl-like alpha-amylase.

[2340] High pH Filter Assay

[2341] Bacillus libraries are plated on a sandwich of cellulose acetate(OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulosefilters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TYagar plates with 10 micro g/ml kanamycin at 37° C. for at least 21hours. The cellulose acetate layer is located on the TY agar plate.

[2342] Each filter sandwich is specifically marked with a needle afterplating, but before incubation in order to be able to localize positivevariants on the filter and the nitrocellulose filter with bound variantsis transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 andincubated at room temperature (can be altered from 10-60° C.) for 15min. The cellulose acetate filters with colonies are stored on theTY-plates at room temperature until use. After incubation, residualactivity is detected on plates containing 1% agarose, 0.2% starch inglycin-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulosefilters are marked the same way as the filter sandwich and incubated for2 hours at room temperature. After removal of the filters the assayplates are stained with 10% Lugol solution. Starch degrading variantsare detected as white spots on dark blue background and then identifiedon the storage plates. Positive variants are rescreened twice under thesame conditions as the first screen.

[2343] Low Calcium Filter Assay

[2344] Bacillus libraries are plated on a sandwich of cellulose acetate(OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulosefilters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TYagar plates with a relevant antibiotic, e.g., kanamycin orchlo-amphenicol, at 37° C. for at least 21 hours. The cellulose -acetatelayer is located on the TY agar plate.

[2345] Each filter sandwich is specifically marked with a needle afterplating, but before incubation in order to be able to localize positivevariants on the filter and the nitrocellulose filter with bound variantsis transferred to a container with carbonate/bicarbonate buffer pH8.5-10 and with different EDTA concentrations (0.001 mM-100 mM). Thefilters are incubated at room temperature for 1 hour. The celluloseacetate filters with colonies are stored on the TY-plates at roomtemperature until use. After incubation, residual activity is detectedon plates containing 1% agarose, 0.2% starch in carbonate/bicarbonatebuffer pH 8.5-10. The assay plates with nitrocellulose filters aremarked the same way as the filter sandwich and incubated for 2 hours atroom temperature. After removal of the filters the assay plates arestained with 10% Lugol solution. Starch degrading variants are detectedas white spots on dark blue background and then identified on thestorage plates. Positive variants are rescreened twice under the sameconditions as the first screen.

[2346] Low pH Filter Assay

[2347] Bacillus libraries are plated on a sandwich of cellulose acetate(OE 67, Schleicher & Schuell, Dassel, Germany)—and nitrocellulosefilters (Protran-Ba 85, Schleicher & Schuell, Dasseli Germany) on TYagar plates with 10 micro g/ml chloramphenicol at 37° C. for at least 21hours. The cellulose acetate layer is located on the TY agar plate.

[2348] Each filter sandwich is specifically marked with a needle afterplating, but before incubation in order to be able to localize positivevariants on the filter, and the nitrocellulose filter with boundvariants is transferred to a container with citrate buffer, pH 4.5 andincubated at 80° C. for 20 minutes (when screening for variants in thewild type backbone) or 85° C. for 60 minutes (when screening forvariants of the parent alpha-amylase). The cellulose acetate filterswith colonies are stored on the TY-plates at room temperature until use.After incubation, residual activity is detected on assay platescontaining 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The assayplates with nitrocellulose filters are marked the same way as the filtersandwich and incubated for 2 hours at 50° C. After removal of thefilters the assay plates are stained with 10% Lugol solution. Starchdegrading variants are detected as white spots on dark blue backgroundand then identified on the storage plates. Positive variants arere-screened twice under the same conditions as the first screen.

[2349] Secondary Screening

[2350] Positive transformants after rescreening are picked from thestorage plate and tested in a secondary plate assay. Positivetransformants are grown for 22 hours at 37° C. in 5 mlLB+chloramphenicol. The Bacillus culture of each positive transformantand as a control a clone expressing the corresponding backbone areincubated in citrate buffer, pH 4.5 at 90° C. and samples are taken at0, 10, 20, 30, 40, 60 and 80 minutes. A 3 micro liter sample is spottedon an assay plate. The assay plate is stained with 10% Lugol solution.Improved variants are seen as variants with higher residual activity(detected as halos on the assay plate) than the backbone. The improvedvariants are determined by nucleotide sequencing.

[2351] Stability Assay of Unpurified Variants

[2352] Bacillus cultures expressing the variants to be analysed aregrown for 21 hours at 37° C. in 10 ml LB+chloramphenicol. 800 microliter culture is mixed with 200 micro I citrate buffer, pH 4.5. A numberof 70 micro I aliquots corresponding to the number of sample time pointsare made in PCR tubes and incubated at 70° C. or 90° C. for various timepoints (typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine.The 0 min sample is not incubated at high temperature. Activity in thesample is measured by transferring 20 micro I to 200 micro I of thealpha-amylase PNP-G₇ substrate MPR3 ((Boehringer Mannheim Cat. no.1660730) as described below under “Assays for Alpha-Amylase Activity”.Results are plotted as percentage activity (relative to the 0 timepoint) versus time, or stated as percentage residual activity afterincubation for a certain period of time.

[2353] Fermentation and Purification of Alpha-Amylase Variants

[2354] A B. subtilis strain harbouring the relevant expression plasmidis streaked on a LB-agar plate with 10 micro g/ml kanamycin from −80° C.stock, and grown overnight at 37° C. The colonies are transferred to 100ml PS-1 media supplemented with 10 micro g/ml chloamphinicol in a 500 mlshaking flask.

[2355] Composition of PS-1 medium Pearl sugar 100 g/l Soy Bean Meal 40g/l Na₂HPO₄, 12 H₂O 10 g/l Pluronic ™ PE 6100 0.1 g/l CaCO₃ 5 g/l

[2356] The culture is shaken at 37° C. at 270 rpm for 5 days.

[2357] Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on a UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied ona S-sepharose F.F. and elution is carried out by step elution with 0.2MNaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions that contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% W/vol.active coal in 5 minutes.

[2358] Stability Determination of Purified Variants

[2359] All stability trials of purified variants are made using the sameset up. The method is as follows:

[2360] The enzyme is incubated under the relevant conditions (1-4).Samples are taken at various time points, e.g., after 0, 5, 10, 15 and30 minutes and diluted 25 times (same dilution for all taken samples) inassay buffer (0.1M 50 mM Britton buffer pH 7.3) and the activity ismeasured using the Phadebas assay (Pharmacia) under standard conditionspH 7.3, 37° C.

[2361]

[2362] The activity measured before incubation (0 minutes) is used asreference (100%). The decline in percent is calculated as a function ofthe incubation time. The table shows the residual activity after, e.g.,30 minutes of incubation.

[2363] Specific Activity Determination

[2364] The specific activity is determined using the Phadebas® assay(Pharmacia) as activity/mg enzyme. The manufactures instructions arefollowed (see also below under “Assay for Alpha-Amylase Activity).

[2365] Determination of Isoelectric P Int

[2366] The pI is determined by isoelectric focusing (ex: Pharmacia,Ampholine, pH 3.5-9.3).

[2367] Stability Determination

[2368] The amylase stability is measured using the method as follows:

[2369] The enzyme is incubated under the relevant conditions. Samplesare taken at various time points, e.g., after 0, 5, 10, 15 and 30minutes and diluted 25 times (same dilution for all taken samples) inassay buffer (0.1 μM 50 mM Britton buffer pH 7.3) and the activity ismeasured using the Phadebas assay (Pharmacia) under standard conditionspH 7.3, 37° C.

[2370] The activity measured before incubation (0 minutes) is used asreference (100%). The decline in percent is calculated as a function ofthe incubation time. The table shows the residual activity after, e.g.,30 minutes of incubation.

[2371] Measurement of the Calcium- and pH-Dependent Stability

[2372] Normally industrial liquefaction processes runs using pH 6.0-6.2as liquefaction pH and an addition of 40 ppm free calcium in order toimprove the stability at 95° C.-105° C. Some of the herein proposedsubstitutions have been made in order to improve the stability at

[2373] 1. lower pH than pH 6.2 and/or

[2374] 2. at free calcium levels lower than 40 ppm free calcium.

[2375] Two different methods can be used to measure the alterations instability obtained by the different substitutions in the alpha-amylasein question:

[2376] Method 1. One assay which measures the stability at reduced pH,pH 5.0, in the presence of 5 ppm free calcium.

[2377] 10 micro g of the variant are incubated under the followingconditions: A 0.1 M acetate solution, pH adjusted to pH 5.0, containing5 ppm calcium and 5% w/w common corn starch (free of calcium).Incubation is made in a water bath at 95° C. for 30 minutes.

[2378] Method 2. One assay, which measure the stability in the absenceof free calcium and where the pH is maintained at pH. 6.0. This assaymeasures the decrease in calcium sensitivity:

[2379] 10 micro g of the variant were incubated under the followingconditions: A 0.1 M acetate solution, pH adjusted to pH 6.0, containing5% w/w common corn starch (free of calcium). Incubation was made in awater bath at 95° C. for 30 minutes.

[2380] Oxidation Stability Determination

[2381] Raw filtered culture broths with different vatiants of theinvention are diluted to an amylase activity of 100 KNU/ml (definedabove) in 50 mM of a Britton-Robinson buffer at pH 9.0 and incubated at40° C. Subsequently H₂O₂ is added to a concentration of 200 mM, and thepH value is re-adjusted to 9.0. The activity is now measured after 15seconds and after 5, 15, and 30 minutes. The absorbance of the resultingblue solution, measured spectrophotometrically at 620 nm, is a functionof the alpha-amylase activity.

[2382] Washing Performance

[2383] Washing performance is evaluated by washing soiled test swatchesfor 15 and 30 minutes at 25° C. and 40° C., respectively; at a pH in therange from 9-10.5; water hardness in the range from 6 to 15° dH; Ca:Mgratio of from 2:1 to 4:1, in different detergent solutions (see above asdescribed above in the Materials section) dosed from 1 to 5 g/l,such as3 g/l, dependent on the detergent with the alpha-amylase variant inquestion.

[2384] The recombinant alpha-amylase variant is added to the detergentsolutions at concentrations of for instance 0.01-5 mg/l. The testswatches aree soiled with orange rice starch (CS-28 swatches availablefrom CFT, Center for Test Material, Holland).

[2385] After washing, the swatches are evaluated by measuring theremission at 460 nm using an Elrepho Remission Spectrophotometer. Theresults are expressed as DeltaR=remission (° R of the swatch washed withthe alpha-amylase minus the remission of a swatch washed at the sameconditions without the alpha-amylase.

[2386] Assays for Alpha-Amylase Activity

[2387] 1. Phadebas Assay

[2388] Alpha-amylase activity is determined by a method employingPhadebas® tablets as substrate. Phadebas tablets (Phadebas® AmylaseTest, supplied by Pharmacia Diagnostic) contain a cross-linked insolubleblue-colored starch polymer, which has been mixed with bovine serumalbumin and a buffer substance and tabletted.

[2389] For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric add, 50 mM boricacid, 0.1 mM CaCl₂, pH adjusted to the valueof interest with NaOH). The test is performed in a water bath at thetemperature of interest. The alpha-amylase to be tested is diluted in xml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylase solutionis added to the 5 ml 50 mM Britton-Robinson buffer. The starch ishydrolyzed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

[2390] It is important that the measured 620 nm absorbance after 10 or15 minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyze a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

[2391] 2. Alternative Method

[2392] Alpha-amylase activity is determined by a method employing thePNP-G₇ substrate. PNPG₇ which is a abbreviation forp-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide whichcan be deaved by an endo-amylase. Following the cleavage, thealpha-Glucosidase included in the kit digest the substrate to liberate afree PNP molecule which has a yellow colour and thus can be measured byvisible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G₇substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim(cat. No.1054635).

[2393] To prepare the reagent solution 10 ml of substrate/buffersolution is added to 50 ml enzyme/buffer solution as recommended by themanufacturer. The assay is performed by transferring 20 micro I sampleto a 96 well microtitre plate and incubating at 25° C. 200 micro Ireagent solution pre-equilibrated to 25° C. is added. The solution ismixed and pre-incubated 1 minute and absorption is measured every 30sec. over 4 minutes at OD 405 nm in an ELISA reader.

[2394] The slope of the time dependent absorption-curve is directlyproportional to the activity of the alpha-amylase in question under thegiven set of conditions.

[2395] Determination of LAS Sensitivity

[2396] The variant is incubated with different concentrations of LAS(linear alkyl benzene sulphonate; Nansa 1169/P) for 10 minutes at 40° C.

[2397] The residual activity is determined using the Phadebas® assaymethod or the alternative method employing the PNP-G₇ substrate.

[2398] LAS is diluted in 0.1 M phosphate buffer pH 7.5.

[2399] The following concentrations are used:

[2400] 500 ppm, 250 ppm, 100 ppm, 50 ppm, 25 ppm, and 10 ppm on no LAS.

[2401] The variant is diluted in the different LAS buffers toconcentration of 0.01-5 mg/l in a total volume of 10 ml and incubatedfor 10 minutes in a temperature controlled water bath. The incubation isstopped by transferring a small aliquat into cold assay buffer. It isimportant that during activity measurement the LAS concentration isbelow 1 ppm, in order not to affect the activity meassurement.

[2402] Then the residual activity is determined in duplicate using theabove mentioned Phadebas® assay or alternative method.

[2403] The activity is measured after subtraction of the blank.

[2404] The activity with no LAS is 100%.

EXAMPLES Example 1

[2405] Construction of Variants of the Invention and Determination ofAltered Properties

[2406] The below listed variants are constructed as described in EXAMPLE1 of WO 00/29560 (from Novozymes A/S) in the parent Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 8.

[2407] G5A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2408] T6A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2409] G36A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2410] I37A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2411] T38A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2412] A39R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2413] I42A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2414] A45R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2415] K47A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2416] D63A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2417] E66A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2418] Q69A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2419] K70A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2420] G71A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2421] T72A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2422] R74A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2423] T75A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2424] K76A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2425] T79A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2426] E82A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2427] L83A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2428] A86R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2429] I87A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2430] S89A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2431] R93A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2432] T12A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2433] E113A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2434] A117R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2435] V120A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2436] A137R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2437] K213A,R,N,D,C,Q,E,G,H,i,L,M,F,P,S,T,W,Y,V;

[2438] G216A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2439] A220R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2440] L223A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2441] L225A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2442] D226A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2443] G227A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2444] R229A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2445] D243A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2446] V245A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2447] F279A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2448] S282A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2449] T311A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2450] V321A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2451] V324A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2452] L352A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2453] T353A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2454] R354A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2455] G357A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2456] V361A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2457] F362A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2458] G364A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2459] G368A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2460] A390R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2461] A395R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2462] G397A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2463] Q399A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2464] H400A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2465] D401A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2466] A425R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2467] D451A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2468] I452A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2469] T453A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2470] G466A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2471] G468A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2472] F470A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2473] H471A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2474] S478A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V

[2475] L7A,R,N,D,C,Q,E,G,H,K,M,P,S,Y,V;

[2476] M8C;

[2477] Q9A,R,N,D,C,G,H,M,P,S,T,W,Y,V;

[2478] F11A,N,D,C,Q,G,H,I,L,M,P,S,T,W,Y,V;

[2479] E12A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2480] G19A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2481] H21A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2482] W22A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2483] L25A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2484] L32A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2485] V40A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2486] W41A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2487] Y46A,R,D,C,G,K,M,P,W;

[2488] G48R,N,D,C,Q,E,H,K,M,F,P,W,Y;

[2489] G55A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2490] G57R,N,D,C,Q,E,H,K,M,P,W;

[2491] A58R,N,D,C,Q,E,G,H,K,M,S,T,W,Y;

[2492] D60A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2493] Y77A,R,D,C,G,K,M,P,W;

[2494] 195A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2495] V97A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2496] Y98A,R,D,C,G,K,M,P,W;

[2497] G99R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2498] D100A,R,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2499] V101A,N,C,Q,G,I,L,M,P,S,T,W,Y;

[2500] V102N,D,C,Q,E,H,I,L,M,F,P,W,Y;

[2501] I103A,N,D,C,Q,E,G,M,P,S,W,Y;

[2502] H105A,N,C,Q,G,I,L,M,P,S,T,Y,V;

[2503] G107R,N,D,Q,E,H,K,M,F,P,W,Y;

[2504] V115R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

[2505] V118R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

[2506] I135A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2507] T139A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2508] F141A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2509] F143A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

[2510] S151A,R,N,D,C,Q,E,G,H,K,M,P,T,Y,V;

[2511] H159A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[2512] F160A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2513] D161A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2514] G162A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2515] T163A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2516] D166A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2517] Y175A,R,D,C,G,K,M,P,W;

[2518] F177A,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;

[2519] D183A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2520] V186A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2521] S187A,R,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[2522] N192A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2523] A199R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2524] D200A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2525] D202A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2526] Y203A,R,D,C,G,K,M,P;

[2527] V208A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y;

[2528] I212A,N,C,Q,G,H,M,P,S,T,V;

[2529] W215A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2530] Y219A,R,D,C,G,K,M,P,W;

[2531] F228A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2532] L230A,R,N,D,C,Q,E,G,M,P,S,T,W,Y,V;

[2533] V233R,N,C,Q,E,G,H,I,K,M,P,S,T,W,Y;

[2534] I236A,C,Q,G,H,M,P,S,T,V;

[2535] F238A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;

[2536] F240A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2537] L241A,N,C,Q,G,H,P,S,T,V;

[2538] W244A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2539] V248A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2540] M256C;

[2541] T258A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2542] V259A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y;

[2543] A260R,N,D,C,Q,E,H,I,L,K,M,F,P,T,W,Y,V;

[2544] Y262A,R,D,C,G,K,M,P,W;

[2545] L270A,N,C,Q,G,I,M,F,P,S,T,W,Y,V;

[2546] Y273A,R,D,C,G,K,M,P;

[2547] L274A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2548] T277A,R,N,D,C,Q,E,G,H,K,M,P,S,W,Y,V;

[2549] H281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;

[2550] V283A,R,N,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y;

[2551] F284A,R,N,D,C,Q,E,G,I,L,K,M,P,S,T,Y,V;

[2552] D285A,R,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2553] V286A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y;

[2554] P287R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2555] L288A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2556] H289A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2557] F292A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2558] A295R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2559] S296A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2560] M304C;

[2561] L307A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2562] V312A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y;

[2563] V313A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2564] S320R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2565] T322R,N,D,C,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

[2566] F323A,R,N,D,C,Q,E,G,I,L,K,M,P,S,T,W,Y,V;

[2567] D325A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2568] N326A,R,C,Q,E,G,M,P,S,T,W;

[2569] H327A,R,C,G,I,L,K,M,P,S,T,W,Y,V;

[2570] T329A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[2571] P331N,D,C,Q,E,G,H,I,L,M,F,S,T,W,Y,V;

[2572] V339A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2573] F343A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

[2574] K344A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2575] L346A,R,N,D,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2576] A347R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2577] A349R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2578] F350A,R,N,C,Q,G,H,I,L,K,M,P,S,T,Y,V;

[2579] P359R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2580] Q360N,D,G,H,I,L,M,F,P,S,T,W,Y,V;

[2581] T369A,R,N,D,CiQ,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2582] I377A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2583] L380A,R,N,D,C,Q,E,G,H,K,P,S,W,Y,V;

[2584] I387A,R,N,D,C,Q,E,G,H,L,K,M,P,S,T,W,Y,V;

[2585] V409N,C,Q,E,G,H,M,P,S,T,W,Y;

[2586] G410A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2587] W411A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2588] T412R,N,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2589] G423A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2590] L424A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2591] A426R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2592] L427A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;

[2593] I428A,N,D,Q,E,G,H,M,F,P,S,W,Y,V;

[2594] T429A,R,N,D,C,Q,E,G,H,I ,L,K,M,F,P,S,W,Y,V;

[2595] D430A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2596] M438C;

[2597] V440R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2598] G441A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2599] W449A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2600] I462A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;

[2601] V472A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2602] V477A,R,N,D,C Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2603] I479A,R,N,D,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

[2604] Y480A,R,D,C,G,K,M,P,W;

[2605] V481A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y.

[2606] A1R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2607] N2R,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2608] L3A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y;

[2609] N4A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2610] W13R,N,D,C,Q,E,G,H,K,M,P,S,T;

[2611] Y14A,R,D,C,G,K,M,P,W;

[2612] P16R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2613] N17A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2614] D18A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2615] Q20A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2616] R23A,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

[2617] R24A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2618] Q26A,D,C,E,G,H,I,L,M,F,P,S,T,W,V;

[2619] E34A,R,N,C,Q,G,H,I,L,K,M,F,P,T,W,Y,V;

[2620] H35A,R,N,D,C,Q,E,G,K,M,F,P,S,T,W,Y,V;

[2621] T49A,C,G,H,P;

[2622] S50A,R,N,C,Q,E,G,H,K,M,F,P,W;

[2623] Q51A,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2624] A52R,D,C,Q,E,G,H,K,P;

[2625] D53A,C,G,H,K,M,P;

[2626] L61A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,Y;

[2627] Y62A,R,D,C,G,K,M,P;

[2628] F67A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;

[2629] H68A,R,D,C,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2630] V73A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y;

[2631] Q84A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2632] S85A,R,N,C,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2633] K88A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2634] H91A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2635] S92A,R,N,D,C,Q,E,G,H,I,L,M,F,P,T,W,Y,V;

[2636] N96A,R,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2637] K106A,N,D,C,Q,E,G,H,I,L,M,P,S,T,Y,V;

[2638] G108R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2639] D114A,N,C,Q,E,G,H,K,F,P,S,T,W,Y;

[2640] T116A,R,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;

[2641] E119A,R,N,D,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2642] D121A,R,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2643] P122R,N,Q,G,H,I,L,M,F,S,T,W,Y,V;

[2644] A123N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2645] D124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2646] R125N,Q,E,G,I,K,M,F,S,T,W,Y;

[2647] N126Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2648] R127A,N,D,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2649] V128A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,W,Y;

[2650] I129A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

[2651] S130A,R,N,D,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[2652] G131A,R,N,D,C,Q,H,I,L,K,M,F,P,S,T,W,Y,V;

[2653] E132R,N,D,C,Q,G,H,I,L,K,M,F,S,W,Y;

[2654] H133R,N,D,C,M,T,W,V;

[2655] L134A,N,D,C,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2656] K136A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2657] W138R,N,D,Q,E,G,I,K,M,P,S,T,V;

[2658] G145A,R,N,D,C,Q,E,H,I,L,K,M,P,S,T,Y,V;

[2659] G147A,R,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;

[2660] S148A,R,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2661] T149A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,W,Y,V;

[2662] Y150A,D,C,G,M,P,W;

[2663] D152A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2664] F153A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2665] K154A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2666] W155A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2667] H156A,C,Q,E,G,I,L,M,F,P,S,T,W,V;

[2668] W157R,I,L,M,F,P,S,T,Y,V;

[2669] Y158R,M,P,W;

[2670] D164R,I,L,M,F,P,S,T,W,Y,V;

[2671] W165A,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,Y,V;

[2672] E167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2673] S168A,R,N,D,C,Q,E,G,H,I,L,K,M,F,T,W,V;

[2674] R169A,N,D,C,Q.E,G,H,M,P,S,W,Y,V;

[2675] K170A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2676] L171A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

[2677] N172A,D,C,Q,E,G,I,L,M,F,P,T,W,Y,V;

[2678] R173A,N,D,C,Q,E,G,H,M,P,S,W,Y,V;

[2679] K176A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2680] G179D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;

[2681] K180A,G,I,L,M,F,P,W,Y,V;

[2682] A181G;

[2683] W182A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2684] W184I,L,M,F,P,W,Y,V;

[2685] E185R,I,L,M,F,P,S,T,W,Y,V;

[2686] N188A,R,Q,G,H,L,M,F,W,V;

[2687] E189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

[2688] G191A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[2689] Y193A,R,G,M,P,W;

[2690] L196A,N,Q,G,H,I,M,P,S,T,W,V;

[2691] Y198A,R,G,M,P,W;

[2692] D204R,L,M,F,P,T,W,Y,V;

[2693] H205A,G,I,L,M,F,P,W,Y,V;

[2694] P206R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2695] A209R,P,S,W,Y;

[2696] A21 OR,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[2697] R214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;

[2698] T217A,R,N,D,C,Q,G,H,I,L,M,F,P,S,W,Y;

[2699] W218A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2700] N221A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2701] E222A,R,N,D,C,G,H,I,L,K,M,F,P,S,W,Y,V;

[2702] K234A,D,C,G,H,I,M,F,P,S,T,W,Y,V;

[2703] H235A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;

[2704] K237A,G,H,I,L,M,F,W,Y,V;

[2705] S239G,H,I,L,M,F,P,T,Y,V;

[2706] R242G,I,L,M,F,S,T,W,Y,V;

[2707] N246A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2708] H247R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[2709] R249A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2710] E250A,R,N,D,C,H,I,L,K,M,P,T,W,Y,V;

[2711] K251 R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2712] T252A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2713] G253R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;

[2714] K254A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2715] E255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;

[2716] F257A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;

[2717] E261A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2718] W263A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,Y,V;

[2719] N265C,Q,E,H,I,L,M,F,P,W;

[2720] D266A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2721] L267A,R,N,D,C,Q,E,G,H,K,P,S,T,W,Y,V;

[2722] G268A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2723] A269N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2724] E271A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2725] N272A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2726] N275A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2727] K276A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2728] N278A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2729] N280A,R,D,C,E,G,H,I,L,K,M,F,P,W,Y,V;

[2730] Y290W;

[2731] Q291A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V;

[2732] H293A,R,N,G,I,L,M,P,S,T,W,V;

[2733] A294R,N,Q,G,H,I,L,M,F,P,S,T,W,Y;

[2734] T297A,G,H,I,L,M,F,P,W,Y,V;

[2735] Q298G,H,I,L,M,F,P,S,T,W,Y,V;

[2736] G299A,H,M,P,S,T;

[2737] G300A,C,H,I,L,M,F,P,T,W,Y,V;

[2738] G301N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[2739] Y302R,M,P,W;

[2740] D303R,I,L,M,F,P,S,T,W,Y,V;

[2741] R305G,I,L,M,F,P,S,T,W,Y,V;

[2742] K306Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2743] L308A,R,N,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;

[2744] N309Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2745] G310A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2746] S314A,D,C,E,G,I,L,M,F,P,W,Y,V;

[2747] K315A,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2748] H316A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[2749] P317R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2750] L318A,R,N,D,C,Q,E,G,H,I,K,P,S,W,Y,V;

[2751] K319A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2752] D328A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2753] G332A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2754] Q333A,N,D,C,G,I,M,F,P,S,T,Y,V;

[2755] S334R,N,C,Q,E,G,H,K,M,F,P,W,Y;

[2756] L335R,D,C,Q,E,H,I,K,M,F,P,W,Y,V;

[2757] E336A,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2758] S337A,R,N,C,Q,E,G,H,I,L,M,F,P,T,W,Y,V;

[2759] T338A,R,N,C,Q,G,H,I,L,K,M,F,P,S,W,Y,V;

[2760] Q340I,L,M,F,P,S,T,W,Y,V;

[2761] T341A,R,D,C,G,H,I,L,K,M,F,W,Y,V;

[2762] W342R,I,L,M,F,P,S,T,Y,V;

[2763] P345R,N,D,C,Q,E,G,H,I,L,K,M,F,T,W,Y,V;

[2764] E355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2765] Y358A,R,D,C,G,K,M,P,W;

[2766] Y363A,R,D,C,G,K,M,P,W;

[2767] K370A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2768] G371A,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2769] S373A,R,N,D,C,Q,E,G,H,I,L,K,M,F,T,W,Y,V;

[2770] Q374A,N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2771] R375A,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,V;

[2772] E376A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2773] P378R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2774] A379R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2775] K381A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2776] K389A,D,C,G,H,M,F,P,S,T,W,Y,V;

[2777] Q393A,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2778] Y394A,R,D,C,G,K,M,P,W;

[2779] Y396A,R,D,C,G,K,M,P,W;

[2780] A398R,N,D,C,Q,E,G,H,I,L,M,F,W,Y,V;

[2781] Y402R,C,G,K,M,P,W;

[2782] F403A,R,N,Q,G,H,I,L,M,P,S,T,W,Y,V;

[2783] D404R,I,L,M,F,P,S,T,W,Y,V;

[2784] H405R,G,I,L,M,F,P,W,Y,V;

[2785] H406A,R,G,I,M,F,P,Y,V;

[2786] D407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2787] I408A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;

[2788] R413A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2789] E414A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2790] G415A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2791] D416A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2792] S417A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;

[2793] S418A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2794] V419A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y;

[2795] A420N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;

[2796] N421A,R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2797] S422A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2798] G431A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[2799] P432G,H,I,L,M,F,S,T,W,Y,V;

[2800] G433A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[2801] G434A,R,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[2802] A435Q,G,H,I,L,M,F,P,T,W,Y,V;

[2803] K436A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2804] R437A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2805] Y439A,R,D,C,G,K,M,P,W;

[2806] R442A,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2807] Q443A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2808] N444A,C,G,H,I,L,M,F,P,S,T,W,Y,V;

[2809] A445R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2810] G446A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2811] E447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;

[2812] T448A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;

[2813] H450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;

[2814] G454A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2815] N455A,R,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2816] R456A,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;

[2817] S457A,R,N,D,C,Q,E,G,H,I,L,K,M,F,W,Y,V;

[2818] E458A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2819] P459R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y,V;

[2820] V460A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;

[2821] V461A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y;

[2822] N463A,R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;

[2823] S464A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;

[2824] E465A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2825] W467A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;

[2826] E469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

[2827] N473Q,G,H,I,L,M,F,P,S,T,W,Y,V;

[2828] G474A,R,H,I,L,M,F,P,W,Y,V;

[2829] G475A,N,Q,H,I,L,M,F,P,S,T,W,Y,V;

[2830] S476G,H,I,L,M,F,P,T,W,Y,V;

[2831] Q482A,N,D,C,G,H,I,L,M,F,S,T,W,Y,V;

[2832] R483A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V.

[2833] The variants are tested for altercd substrate specificity,substrate binding, substrate cleavage pattern, thermal stability, pHactivity profile, pH stability profile, stability towards oxidation,Ca²⁺ dependency, reduced LAS sensitivity, reduced and increased pI andimproved wash performance, and specific activity as described in the“Materials & Methods” section above.

Example 2

[2834] Construction of Variants of the Invention and Determination ofAltered Properties

[2835] The below listed variants are constructed as described in EXAMPLE1 of WO 00/37626 (from Novozymes A/S) in the parent Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 8. The alterations ofthe variants are, as specified in the list below, insertion of an aminoacid downstream of the amino acid which occupies the position, ordeletion of the amino acid which occupies the position.

[2836] A1 insertion;

[2837] L3 insertion;

[2838] N4 insertion;

[2839] N17 insertion;

[2840] D18 insertion;

[2841] Q20 insertion;

[2842] R23 insertion;

[2843] R24 insertion;

[2844] D28 insertion;

[2845] Y56 insertion;

[2846] L61 insertion or deletion;

[2847] Y62 insertion;

[2848] F67 insertion or deletion;

[2849] H68 insertion;

[2850] K80 insertion or deletion;

[2851] G81 insertion or deletion;

[2852] Q84 insertion;

[2853] S85 insertion;

[2854] H91 insertion or deletion;

[2855] S92 insertion or deletion;

[2856] K106 insertion or deletion;

[2857] D110 insertion or deletion;

[2858] D114 deletion;

[2859] E119 insertion or deletion;

[2860] D121 insertion;

[2861] P122 insertion;

[2862] A123 insertion;

[2863] D124 insertion;

[2864] R125 insertion;

[2865] N126 insertion;

[2866] R127 insertion;

[2867] I129 insertion;

[2868] G131 insertion;

[2869] L134 insertion;

[2870] K136 insertion;

[2871] N172 insertion;

[2872] E185 insertion;

[2873] L196 insertion or deletion;

[2874] P206 insertion or deletion;

[2875] T217 insertion;

[2876] W218 insertion;

[2877] D231 insertion or deletion;

[2878] A232 insertion or deletion;

[2879] H235 insertion or deletion;

[2880] N246 insertion;

[2881] H247 insertion;

[2882] R249 insertion;

[2883] K251 insertion;

[2884] F257 insertion or deletion;

[2885] N278 insertion;

[2886] G310 insertion or deletion;

[2887] H316 insertion;

[2888] P317 insertion;

[2889] D328 insertion or deletion;

[2890] G332 insertion or deletion;

[2891] E355 insertion or deletion;

[2892] Y358 insertion;

[2893] Y363 insertion;

[2894] Y367 insertion;

[2895] K370 insertion;

[2896] S373 insertion;

[2897] R375 insertion;

[2898] E376 insertion;

[2899] K381 insertion;

[2900] H382 insertion;

[2901] R391 insertion or deletion;

[2902] Y396 insertion;

[2903] R413 insertion or deletion;

[2904] E414 insertion or deletion;

[2905] G415 insertion or deletion;

[2906] D416 insertion;

[2907] S417 insertion;

[2908] S418 insertion;

[2909] V419 insertion;

[2910] A420 insertion;

[2911] N421 insertion;

[2912] S422 insertion or deletion;

[2913] Y439 insertion;

[2914] A445 insertion or deletion;

[2915] G446 insertion or deletion;

[2916] T448 insertion or deletion;

[2917] H450 insertion;

[2918] G454 insertion or deletion;

[2919] N455 insertion;

[2920] E458 insertion;

[2921] P459 insertion;

[2922] V460 insertion;

[2923] V461 insertion;

[2924] N463 insertion;

[2925] S464 insertion;

[2926] E465 insertion;

[2927] W467 insertion;

[2928] L7 insertion or deletion;

[2929] M8 insertion;

[2930] Y10 insertion;

[2931] F11 insertion;

[2932] E12 insertion or deletion;

[2933] M15 insertion;

[2934] G19 insertion;

[2935] H21 insertion;

[2936] W22 insertion;

[2937] L25 insertion;

[2938] V40 insertion or deletion;

[2939] W41 insertion;

[2940] P43 insertion or deletion;

[2941] P44 insertion or deletion;

[2942] Y46 insertion;

[2943] G55 insertion;

[2944] Y59 insertion;

[2945] Y77 insertion;

[2946] G78 insertion or deletion;

[2947] L90 insertion or deletion;

[2948] I95 insertion;

[2949] V97 insertion;

[2950] Y98 insertion;

[2951] G99 insertion;

[2952] D10 insertion;

[2953] V101 insertion;

[2954] V102 insertion;

[2955] H105 insertion or deletion;

[2956] A109 insertion or deletion;

[2957] V115 insertion or deletion;

[2958] V118 insertion or deletion;

[2959] I135 insertion;

[2960] T139 insertion or deletion;

[2961] F141 insertion or deletion;

[2962] Y195 insertion;

[2963] V208 insertion or deletion;

[2964] W215 insertion;

[2965] Y219 insertion;

[2966] I236 insertion or deletion;

[2967] F238 insertion or deletion;

[2968] F240 insertion or deletion;

[2969] W244 insertion;

[2970] V248 insertion;

[2971] M256 insertion;

[2972] T258 insertion or deletion;

[2973] V259 insertion or deletion;

[2974] V312 insertion or deletion;

[2975] V313 insertion or deletion;

[2976] S320 insertion;

[2977] T322 insertion or deletion;

[2978] F323 insertion or deletion;

[2979] D325 insertion or deletion;

[2980] N326 insertion;

[2981] H327 insertion or deletion;

[2982] Q330 insertion or deletion;

[2983] P331 insertion or deletion;

[2984] Y348 insertion;

[2985] A349 insertion or deletion;

[2986] F350 insertion or deletion;

[2987] P359 insertion or deletion;

[2988] Q360 insertion;

[2989] D365 insertion or deletion;

[2990] M366 insertion;

[2991] T369 insertion;

[2992] I377 insertion;

[2993] I384 insertion or deletion;

[2994] L388 insertion or deletion;

[2995] G423 insertion or deletion;

[2996] L424 insertion or deletion;

[2997] M438 insertion;

[2998] G441 insertion or deletion;

[2999] W449 insertion;

[3000] I462 insertion;

[3001] I479 insertion or deletion;

[3002] Y480 insertion;

[3003] V481 insertion or deletion.

[3004] The variants are tested for altered substrate specificity,substrate binding, substrate cleavage pattern, thermal stability, pHactivity profile, pH stability profile, stability towards oxidation,Ca²⁺ dependency, reduced and increased pI and improved wash performance,specific activity as described in the “Materials & Methods” sectionabove.

1 13 1 1455 DNA Bacillus sp. CDS (1)..(1455) SP690 1 cat cat aat gga acaaat ggt act atg atg caa tat ttc gaa tgg tat 48 His His Asn Gly Thr AsnGly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 ttg cca aat gac gggaat cat tgg aac agg ttg agg gat gac gca gct 96 Leu Pro Asn Asp Gly AsnHis Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 aac tta aag agt aaa gggata aca gct gta tgg atc cca cct gca tgg 144 Asn Leu Lys Ser Lys Gly IleThr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 aag ggg act tcc cag aat gatgta ggt tat gga gcc tat gat tta tat 192 Lys Gly Thr Ser Gln Asn Asp ValGly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gat ctt gga gag ttt aac cag aagggg acg gtt cgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys GlyThr Val Arg Thr Lys Tyr Gly 65 70 75 80 aca cgc aac cag cta cag gct gcggtg acc tct tta aaa aat aac ggc 288 Thr Arg Asn Gln Leu Gln Ala Ala ValThr Ser Leu Lys Asn Asn Gly 85 90 95 att cag gta tat ggt gat gtc gtc atgaat cat aaa ggt gga gca gat 336 Ile Gln Val Tyr Gly Asp Val Val Met AsnHis Lys Gly Gly Ala Asp 100 105 110 ggt acg gaa att gta aat gcg gta gaagtg aat cgg agc aac cga aac 384 Gly Thr Glu Ile Val Asn Ala Val Glu ValAsn Arg Ser Asn Arg Asn 115 120 125 cag gaa acc tca gga gag tat gca atagaa gcg tgg aca aag ttt gat 432 Gln Glu Thr Ser Gly Glu Tyr Ala Ile GluAla Trp Thr Lys Phe Asp 130 135 140 ttt cct gga aga gga aat aac cat tccagc ttt aag tgg cgc tgg tat 480 Phe Pro Gly Arg Gly Asn Asn His Ser SerPhe Lys Trp Arg Trp Tyr 145 150 155 160 cat ttt gat ggg aca gat tgg gatcag tca cgc cag ctt caa aac aaa 528 His Phe Asp Gly Thr Asp Trp Asp GlnSer Arg Gln Leu Gln Asn Lys 165 170 175 ata tat aaa ttc agg gga aca ggcaag gcc tgg gac tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Thr Gly LysAla Trp Asp Trp Glu Val Asp 180 185 190 aca gag aat ggc aac tat gac tatctt atg tat gca gac gtg gat atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr LeuMet Tyr Ala Asp Val Asp Met 195 200 205 gat cac cca gaa gta ata cat gaactt aga aac tgg gga gtg tgg tat 672 Asp His Pro Glu Val Ile His Glu LeuArg Asn Trp Gly Val Trp Tyr 210 215 220 acg aat aca ctg aac ctt gat ggattt aga ata gat gca gtg aaa cat 720 Thr Asn Thr Leu Asn Leu Asp Gly PheArg Ile Asp Ala Val Lys His 225 230 235 240 ata aaa tat agc ttt acg agagat tgg ctt aca cat gtg cgt aac acc 768 Ile Lys Tyr Ser Phe Thr Arg AspTrp Leu Thr His Val Arg Asn Thr 245 250 255 aca ggt aaa cca atg ttt gcagtg gct gag ttt tgg aaa aat gac ctt 816 Thr Gly Lys Pro Met Phe Ala ValAla Glu Phe Trp Lys Asn Asp Leu 260 265 270 ggt gca att gaa aac tat ttgaat aaa aca agt tgg aat cac tcg gtg 864 Gly Ala Ile Glu Asn Tyr Leu AsnLys Thr Ser Trp Asn His Ser Val 275 280 285 ttt gat gtt cct ctc cac tataat ttg tac aat gca tct aat agc ggt 912 Phe Asp Val Pro Leu His Tyr AsnLeu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 ggt tat tat gat atg aga aatatt tta aat ggt tct gtg gtg caa aaa 960 Gly Tyr Tyr Asp Met Arg Asn IleLeu Asn Gly Ser Val Val Gln Lys 305 310 315 320 cat cca aca cat gcc gttact ttt gtt gat aac cat gat tct cag ccc 1008 His Pro Thr His Ala Val ThrPhe Val Asp Asn His Asp Ser Gln Pro 325 330 335 ggg gaa gca ttg gaa tccttt gtt caa caa tgg ttt aaa cca ctt gca 1056 Gly Glu Ala Leu Glu Ser PheVal Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350 tat gca ttg gtt ctg acaagg gaa caa ggt tat cct tcc gta ttt tat 1104 Tyr Ala Leu Val Leu Thr ArgGlu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 ggg gat tac tac ggt atccca acc cat ggt gtt ccg gct atg aaa tct 1152 Gly Asp Tyr Tyr Gly Ile ProThr His Gly Val Pro Ala Met Lys Ser 370 375 380 aaa ata gac cct ctt ctgcag gca cgt caa act ttt gcc tat ggt acg 1200 Lys Ile Asp Pro Leu Leu GlnAla Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 cag cat gat tac tttgat cat cat gat att atc ggt tgg aca aga gag 1248 Gln His Asp Tyr Phe AspHis His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 gga aat agc tcc catcca aat tca ggc ctt gcc acc att atg tca gat 1296 Gly Asn Ser Ser His ProAsn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 ggt cca ggt ggt aacaaa tgg atg tat gtg ggg aaa aat aaa gcg gga 1344 Gly Pro Gly Gly Asn LysTrp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 caa gtt tgg aga gatatt acc gga aat agg aca ggc acc gtc aca att 1392 Gln Val Trp Arg Asp IleThr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 aat gca gac gga tggggt aat ttc tct gtt aat gga ggg tcc gtt tcg 1440 Asn Ala Asp Gly Trp GlyAsn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 gtt tgg gtg aagcaa 1455 Val Trp Val Lys Gln 485 2 485 PRT Bacillus sp. 2 His His AsnGly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu ProAsn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn LeuLys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys GlyThr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp LeuGly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 ThrArg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 IleGln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130135 140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu GlnAsn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp TrpGlu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Val Asp Met 195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg AsnTrp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe ArgIle Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg AspTrp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe AlaVal Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn TyrLeu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro LeuHis Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr AspMet Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His ProThr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 GlyGlu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp ThrArg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr IleMet Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly LysAsn Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg ThrGly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser ValAsn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 3 1455DNA Bacillus sp. CDS (1)..(1455) SP722 3 cat cat aat ggg aca aat ggg acgatg atg caa tac ttt gaa tgg cac 48 His His Asn Gly Thr Asn Gly Thr MetMet Gln Tyr Phe Glu Trp His 1 5 10 15 ttg cct aat gat ggg aat cac tggaat aga tta aga gat gat gct agt 96 Leu Pro Asn Asp Gly Asn His Trp AsnArg Leu Arg Asp Asp Ala Ser 20 25 30 aat cta aga aat aga ggt ata acc gctatt tgg att ccg cct gcc tgg 144 Asn Leu Arg Asn Arg Gly Ile Thr Ala IleTrp Ile Pro Pro Ala Trp 35 40 45 aaa ggg act tcg caa aat gat gtg ggg tatgga gcc tat gat ctt tat 192 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr 50 55 60 gat tta ggg gaa ttt aat caa aag ggg acg gttcgt act aag tat ggg 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val ArgThr Lys Tyr Gly 65 70 75 80 aca cgt agt caa ttg gag tct gcc atc cat gcttta aag aat aat ggc 288 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala LeuLys Asn Asn Gly 85 90 95 gtt caa gtt tat ggg gat gta gtg atg aac cat aaagga gga gct gat 336 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys GlyGly Ala Asp 100 105 110 gct aca gaa aac gtt ctt gct gtc gag gtg aat ccaaat aac cgg aat 384 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro AsnAsn Arg Asn 115 120 125 caa gaa ata tct ggg gac tac aca att gag gct tggact aag ttt gat 432 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp ThrLys Phe Asp 130 135 140 ttt cca ggg agg ggt aat aca tac tca gac ttt aaatgg cgt tgg tat 480 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys TrpArg Trp Tyr 145 150 155 160 cat ttc gat ggt gta gat tgg gat caa tca cgacaa ttc caa aat cgt 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg GlnPhe Gln Asn Arg 165 170 175 atc tac aaa ttc cga ggt gat ggt aag gca tgggat tgg gaa gta gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp AspTrp Glu Val Asp 180 185 190 tcg gaa aat gga aat tat gat tat tta atg tatgca gat gta gat atg 624 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Val Asp Met 195 200 205 gat cat ccg gag gta gta aat gag ctt aga agatgg gga gaa tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg TrpGly Glu Trp Tyr 210 215 220 aca aat aca tta aat ctt gat gga ttt agg atcgat gcg gtg aag cat 720 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile AspAla Val Lys His 225 230 235 240 att aaa tat agc ttt aca cgt gat tgg ttgacc cat gta aga aac gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu ThrHis Val Arg Asn Ala 245 250 255 acg gga aaa gaa atg ttt gct gtt gct gaattt tgg aaa aat gat tta 816 Thr Gly Lys Glu Met Phe Ala Val Ala Glu PheTrp Lys Asn Asp Leu 260 265 270 ggt gcc ttg gag aac tat tta aat aaa acaaac tgg aat cat tct gtc 864 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr AsnTrp Asn His Ser Val 275 280 285 ttt gat gtc ccc ctt cat tat aat ctt tataac gcg tca aat agt gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr AsnAla Ser Asn Ser Gly 290 295 300 ggc aac tat gac atg gca aaa ctt ctt aatgga acg gtt gtt caa aag 960 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn GlyThr Val Val Gln Lys 305 310 315 320 cat cca atg cat gcc gta act ttt gtggat aat cac gat tct caa cct 1008 His Pro Met His Ala Val Thr Phe Val AspAsn His Asp Ser Gln Pro 325 330 335 ggg gaa tca tta gaa tca ttt gta caagaa tgg ttt aag cca ctt gct 1056 Gly Glu Ser Leu Glu Ser Phe Val Gln GluTrp Phe Lys Pro Leu Ala 340 345 350 tat gcg ctt att tta aca aga gaa caaggc tat ccc tct gtc ttc tat 1104 Tyr Ala Leu Ile Leu Thr Arg Glu Gln GlyTyr Pro Ser Val Phe Tyr 355 360 365 ggt gac tac tat gga att cca aca catagt gtc cca gca atg aaa gcc 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His SerVal Pro Ala Met Lys Ala 370 375 380 aag att gat cca atc tta gag gcg cgtcaa aat ttt gca tat gga aca 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg GlnAsn Phe Ala Tyr Gly Thr 385 390 395 400 caa cat gat tat ttt gac cat cataat ata atc gga tgg aca cgt gaa 1248 Gln His Asp Tyr Phe Asp His His AsnIle Ile Gly Trp Thr Arg Glu 405 410 415 gga aat acc acg cat ccc aat tcagga ctt gcg act atc atg tcg gat 1296 Gly Asn Thr Thr His Pro Asn Ser GlyLeu Ala Thr Ile Met Ser Asp 420 425 430 ggg cca ggg gga gag aaa tgg atgtac gta ggg caa aat aaa gca ggt 1344 Gly Pro Gly Gly Glu Lys Trp Met TyrVal Gly Gln Asn Lys Ala Gly 435 440 445 caa gtt tgg cat gac ata act ggaaat aaa cca gga aca gtt acg atc 1392 Gln Val Trp His Asp Ile Thr Gly AsnLys Pro Gly Thr Val Thr Ile 450 455 460 aat gca gat gga tgg gct aat ttttca gta aat gga gga tct gtt tcc 1440 Asn Ala Asp Gly Trp Ala Asn Phe SerVal Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg gtg aaa cga 1455 IleTrp Val Lys Arg 485 4 485 PRT Bacillus sp. 4 His His Asn Gly Thr Asn GlyThr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly AsnHis Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg GlyIle Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln AsnAsp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe AsnGln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln LeuGlu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 Val Gln Val Tyr GlyAsp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu AsnVal Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu IleSer Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe ProGly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu TrpTyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala ValLys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr HisVal Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu PheTrp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys ThrAsn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn LeuTyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys LeuLeu Asn Gly Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala ValThr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu GluSer Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu IleLeu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp TyrTyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys IleAsp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val ThrIle 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly SerVal Ser 465 470 475 480 Ile Trp Val Lys Arg 485 5 1548 DNA Bacillusstearothermophilus CDS (1)..(1548) BSG 5 gcc gca ccg ttt aac ggc acc atgatg cag tat ttt gaa tgg tac ttg 48 Ala Ala Pro Phe Asn Gly Thr Met MetGln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 ccg gat gat ggc acg tta tgg accaaa gtg gcc aat gaa gcc aac aac 96 Pro Asp Asp Gly Thr Leu Trp Thr LysVal Ala Asn Glu Ala Asn Asn 20 25 30 tta tcc agc ctt ggc atc acc gct ctttgg ctg ccg ccc gct tac aaa 144 Leu Ser Ser Leu Gly Ile Thr Ala Leu TrpLeu Pro Pro Ala Tyr Lys 35 40 45 gga aca agc cgc agc gac gta ggg tac ggagta tac gac ttg tat gac 192 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly ValTyr Asp Leu Tyr Asp 50 55 60 ctc ggc gaa ttc aat caa aaa ggg acc gtc cgcaca aaa tac gga aca 240 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg ThrLys Tyr Gly Thr 65 70 75 80 aaa gct caa tat ctt caa gcc att caa gcc gcccac gcc gct gga atg 288 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala HisAla Ala Gly Met 85 90 95 caa gtg tac gcc gat gtc gtg ttc gac cat aaa ggcggc gct gac ggc 336 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly GlyAla Asp Gly 100 105 110 acg gaa tgg gtg gac gcc gtc gaa gtc aat ccg tccgac cgc aac caa 384 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser AspArg Asn Gln 115 120 125 gaa atc tcg ggc acc tat caa atc caa gca tgg acgaaa ttt gat ttt 432 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr LysPhe Asp Phe 130 135 140 ccc ggg cgg ggc aac acc tac tcc agc ttt aag tggcgc tgg tac cat 480 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp ArgTrp Tyr His 145 150 155 160 ttt gac ggc gtt gat tgg gac gaa agc cga aaattg agc cgc att tac 528 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys LeuSer Arg Ile Tyr 165 170 175 aaa ttc cgc ggc atc ggc aaa gcg tgg gat tgggaa gta gac acg gaa 576 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp GluVal Asp Thr Glu 180 185 190 aac gga aac tat gac tac tta atg tat gcc gacctt gat atg gat cat 624 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp LeuAsp Met Asp His 195 200 205 ccc gaa gtc gtg acc gag ctg aaa aac tgg gggaaa tgg tat gtc aac 672 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly LysTrp Tyr Val Asn 210 215 220 aca acg aac att gat ggg ttc cgg ctt gat gccgtc aag cat att aag 720 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala ValLys His Ile Lys 225 230 235 240 ttc agt ttt ttt cct gat tgg ttg tcg tatgtg cgt tct cag act ggc 768 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr ValArg Ser Gln Thr Gly 245 250 255 aag ccg cta ttt acc gtc ggg gaa tat tggagc tat gac atc aac aag 816 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp SerTyr Asp Ile Asn Lys 260 265 270 ttg cac aat tac att acg aaa aca gac ggaacg atg tct ttg ttt gat 864 Leu His Asn Tyr Ile Thr Lys Thr Asp Gly ThrMet Ser Leu Phe Asp 275 280 285 gcc ccg tta cac aac aaa ttt tat acc gcttcc aaa tca ggg ggc gca 912 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala SerLys Ser Gly Gly Ala 290 295 300 ttt gat atg cgc acg tta atg acc aat actctc atg aaa gat caa ccg 960 Phe Asp Met Arg Thr Leu Met Thr Asn Thr LeuMet Lys Asp Gln Pro 305 310 315 320 aca ttg gcc gtc acc ttc gtt gat aatcat gac acc gaa ccc ggc caa 1008 Thr Leu Ala Val Thr Phe Val Asp Asn HisAsp Thr Glu Pro Gly Gln 325 330 335 gcg ctg cag tca tgg gtc gac cca tggttc aaa ccg ttg gct tac gcc 1056 Ala Leu Gln Ser Trp Val Asp Pro Trp PheLys Pro Leu Ala Tyr Ala 340 345 350 ttt att cta act cgg cag gaa gga tacccg tgc gtc ttt tat ggt gac 1104 Phe Ile Leu Thr Arg Gln Glu Gly Tyr ProCys Val Phe Tyr Gly Asp 355 360 365 tat tat ggc att cca caa tat aac attcct tcg ctg aaa agc aaa atc 1152 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile ProSer Leu Lys Ser Lys Ile 370 375 380 gat ccg ctc ctc atc gcg cgc agg gattat gct tac gga acg caa cat 1200 Asp Pro Leu Leu Ile Ala Arg Arg Asp TyrAla Tyr Gly Thr Gln His 385 390 395 400 gat tat ctt gat cac tcc gac atcatc ggg tgg aca agg gaa ggg ggc 1248 Asp Tyr Leu Asp His Ser Asp Ile IleGly Trp Thr Arg Glu Gly Gly 405 410 415 act gaa aaa cca gga tcc gga ctggcc gca ctg atc acc gat ggg ccg 1296 Thr Glu Lys Pro Gly Ser Gly Leu AlaAla Leu Ile Thr Asp Gly Pro 420 425 430 gga gga agc aaa tgg atg tac gttggc aaa caa cac gct gga aaa gtg 1344 Gly Gly Ser Lys Trp Met Tyr Val GlyLys Gln His Ala Gly Lys Val 435 440 445 ttc tat gac ctt acc ggc aac cggagt gac acc gtc acc atc aac agt 1392 Phe Tyr Asp Leu Thr Gly Asn Arg SerAsp Thr Val Thr Ile Asn Ser 450 455 460 gat gga tgg ggg gaa ttc aaa gtcaat ggc ggt tcg gtt tcg gtt tgg 1440 Asp Gly Trp Gly Glu Phe Lys Val AsnGly Gly Ser Val Ser Val Trp 465 470 475 480 gtt cct aga aaa acg acc gtttct acc atc gct cgg ccg atc aca acc 1488 Val Pro Arg Lys Thr Thr Val SerThr Ile Ala Arg Pro Ile Thr Thr 485 490 495 cga ccg tgg act ggt gaa ttcgtc cgt tgg acc gaa cca cgg ttg gtg 1536 Arg Pro Trp Thr Gly Glu Phe ValArg Trp Thr Glu Pro Arg Leu Val 500 505 510 gca tgg cct tga 1548 Ala TrpPro 515 6 515 PRT Bacillus stearothermophilus 6 Ala Ala Pro Phe Asn GlyThr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly ThrLeu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu GlyIle Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg SerAsp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe AsnGln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln TyrLeu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr AlaAsp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu TrpVal Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu IleSer Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 ProGly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met AspHis 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp TyrVal Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr ValArg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr TrpSer Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr AspGly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe TyrThr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu MetThr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val ThrPhe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln SerTrp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile LeuThr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr TyrGly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 AspPro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly LysVal 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr IleAsn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser ValSer Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile AlaArg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg TrpThr Glu Pro Arg Leu Val 500 505 510 Ala Trp Pro 515 7 1920 DNA Bacilluslicheniformis CDS (421)..(1872) TERMAMYL 7 cggaagattg gaagtacaaaaataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60 gagacggaaa aatcgtcttaatgcacgata tttatgcaac gttcgcagat gctgctgaag 120 agattattaa aaagctgaaagcaaaaggct atcaattggt aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggctattgaataa atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatatagggaaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaaggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga cgctgttatttgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat ctt aat ggg acgctg atg cag tat ttt gaa tgg tac atg ccc 468 Ala Asn Leu Asn Gly Thr LeuMet Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 aat gac ggc caa cat tggagg cgt ttg caa aac gac tcg gca tat ttg 516 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 gct gaa cac ggt att act gccgtc tgg att ccc ccg gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala ValTrp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 acg agc caa gcg gat gtg ggc tacggt gct tac gac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg gttcgg aca aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val ArgThr Lys Tyr Gly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg atc aaa agtctt cat tcc cgc gac att aac 708 Gly Glu Leu Gln Ser Ala Ile Lys Ser LeuHis Ser Arg Asp Ile Asn 85 90 95 gtt tac ggg gat gtg gtc atc aac cac aaaggc ggc gct gat gcg acc 756 Val Tyr Gly Asp Val Val Ile Asn His Lys GlyGly Ala Asp Ala Thr 100 105 110 gaa gat gta acc gcg gtt gaa gtc gat cccgct gac cgc aac cgc gta 804 Glu Asp Val Thr Ala Val Glu Val Asp Pro AlaAsp Arg Asn Arg Val 115 120 125 att tca gga gaa cac cta att aaa gcc tggaca cat ttt cat ttt ccg 852 Ile Ser Gly Glu His Leu Ile Lys Ala Trp ThrHis Phe His Phe Pro 130 135 140 ggg cgc ggc agc aca tac agc gat ttt aaatgg cat tgg tac cat ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys TrpHis Trp Tyr His Phe 145 150 155 160 gac gga acc gat tgg gac gag tcc cgaaag ctg aac cgc atc tat aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg LysLeu Asn Arg Ile Tyr Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaagtt tcc aat gaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu ValSer Asn Glu Asn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac atcgat tat gac cat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile AspTyr Asp His Pro Asp Val 195 200 205 gca gca gaa att aag aga tgg ggc acttgg tat gcc aat gaa ctg caa 1092 Ala Ala Glu Ile Lys Arg Trp Gly Thr TrpTyr Ala Asn Glu Leu Gln 210 215 220 ttg gac ggt ttc cgt ctt gat gct gtcaaa cac att aaa ttt tct ttt 1140 Leu Asp Gly Phe Arg Leu Asp Ala Val LysHis Ile Lys Phe Ser Phe 225 230 235 240 ttg cgg gat tgg gtt aat cat gtcagg gaa aaa acg ggg aag gaa atg 1188 Leu Arg Asp Trp Val Asn His Val ArgGlu Lys Thr Gly Lys Glu Met 245 250 255 ttt acg gta gct gaa tat tgg cagaat gac ttg ggc gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln AsnAsp Leu Gly Ala Leu Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aatcat tca gtg ttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn HisSer Val Phe Asp Val Pro Leu 275 280 285 cat tat cag ttc cat gct gca tcgaca cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser ThrGln Gly Gly Gly Tyr Asp Met 290 295 300 agg aaa ttg ctg aac ggt acg gtcgtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 gtt aca ttt gtc gat aac catgat aca cag ccg ggg caa tcg ctt gag 1428 Val Thr Phe Val Asp Asn His AspThr Gln Pro Gly Gln Ser Leu Glu 325 330 335 tcg act gtc caa aca tgg tttaag ccg ctt gct tac gct ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe LysPro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 aca agg gaa tct gga tac cctcag gtt ttc tac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro GlnVal Phe Tyr Gly Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgcgaa att cct gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg GluIle Pro Ala Leu Lys His Lys Ile 370 375 380 gaa ccg atc tta aaa gcg agaaaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala Arg LysGln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 gat tat ttc gac cac catgac att gtc ggc tgg aca agg gaa ggc gac 1668 Asp Tyr Phe Asp His His AspIle Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 agc tcg gtt gca aat tcaggt ttg gcg gca tta ata aca gac gga ccc 1716 Ser Ser Val Ala Asn Ser GlyLeu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 ggt ggg gca aag cga atgtat gtc ggc cgg caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met TyrVal Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 tgg cat gac att acc ggaaac cgt tcg gag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly AsnArg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga gag tttcac gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe HisVal Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 gtt caa aga tagaagagcagag aggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg tttatttt1920 8 483 PRT Bacillus licheniformis 8 Ala Asn Leu Asn Gly Thr Leu MetGln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp ArgArg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr AlaVal Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val GlyTyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys GlyThr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser AlaIle Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val ValIle Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr AlaVal Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly GluHis Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg GlySer Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 AspGly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe SerPhe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr GlyLys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu GlyAla Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser ValPhe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr GlnGly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val ValSer Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn HisAsp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr TrpPhe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser GlyTyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly AspSer Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro IleLeu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 AspTyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser IleTyr 465 470 475 480 Val Gln Arg 9 2084 DNA Bacillus amyloliquefaciensCDS (343)..(1794) BAN 9 gccccgcaca tacgaaaaga ctggctgaaa acattgagcctttgatgact gatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaa gaagaagaccataaaaatac cttgtctgtc 120 atcagacagg gtatttttta tgctgtccag actgtccgctgtgtaaaaat aaggaataaa 180 ggggggttgt tattatttta ctgatatgta aaatataatttgtataagaa aatgagaggg 240 agaggaaaca tgattcaaaa acgaaagcgg acagtttcgttcagacttgt gcttatgtgc 300 acgctgttat ttgtcagttt gccgattaca aaaacatcag ccgta aat ggc acg 354 Val Asn Gly Thr 1 ctg atg cag tat ttt gaa tgg tatacg ccg aac gac ggc cag cat tgg 402 Leu Met Gln Tyr Phe Glu Trp Tyr ThrPro Asn Asp Gly Gln His Trp 5 10 15 20 aaa cga ttg cag aat gat gcg gaacat tta tcg gat atc gga atc act 450 Lys Arg Leu Gln Asn Asp Ala Glu HisLeu Ser Asp Ile Gly Ile Thr 25 30 35 gcc gtc tgg att cct ccc gca tac aaagga ttg agc caa tcc gat aac 498 Ala Val Trp Ile Pro Pro Ala Tyr Lys GlyLeu Ser Gln Ser Asp Asn 40 45 50 gga tac gga cct tat gat ttg tat gat ttagga gaa ttc cag caa aaa 546 Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu GlyGlu Phe Gln Gln Lys 55 60 65 ggg acg gtc aga acg aaa tac ggc aca aaa tcagag ctt caa gat gcg 594 Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser GluLeu Gln Asp Ala 70 75 80 atc ggc tca ctg cat tcc cgg aac gtc caa gta tacgga gat gtg gtt 642 Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr GlyAsp Val Val 85 90 95 100 ttg aat cat aag gct ggt gct gat gca aca gaa gatgta act gcc gtc 690 Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp ValThr Ala Val 105 110 115 gaa gtc aat ccg gcc aat aga aat cag gaa act tcggag gaa tat caa 738 Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser GluGlu Tyr Gln 120 125 130 atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc cgtgga aac acg tac 786 Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg GlyAsn Thr Tyr 135 140 145 agt gat ttt aaa tgg cat tgg tat cat ttc gac ggagcg gac tgg gat 834 Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly AlaAsp Trp Asp 150 155 160 gaa tcc cgg aag atc agc cgc atc ttt aag ttt cgtggg gaa gga aaa 882 Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg GlyGlu Gly Lys 165 170 175 180 gcg tgg gat tgg gaa gta tca agt gaa aac ggcaac tat gac tat tta 930 Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly AsnTyr Asp Tyr Leu 185 190 195 atg tat gct gat gtt gac tac gac cac cct gatgtc gtg gca gag aca 978 Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp ValVal Ala Glu Thr 200 205 210 aaa aaa tgg ggt atc tgg tat gcg aat gaa ctgtca tta gac ggc ttc 1026 Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu SerLeu Asp Gly Phe 215 220 225 cgt att gat gcc gcc aaa cat att aaa ttt tcattt ctg cgt gat tgg 1074 Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser PheLeu Arg Asp Trp 230 235 240 gtt cag gcg gtc aga cag gcg acg gga aaa gaaatg ttt acg gtt gcg 1122 Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu MetPhe Thr Val Ala 245 250 255 260 gag tat tgg cag aat aat gcc ggg aaa ctcgaa aac tac ttg aat aaa 1170 Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu GluAsn Tyr Leu Asn Lys 265 270 275 aca agc ttt aat caa tcc gtg ttt gat gttccg ctt cat ttc aat tta 1218 Thr Ser Phe Asn Gln Ser Val Phe Asp Val ProLeu His Phe Asn Leu 280 285 290 cag gcg gct tcc tca caa gga ggc gga tatgat atg agg cgt ttg ctg 1266 Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr AspMet Arg Arg Leu Leu 295 300 305 gac ggt acc gtt gtg tcc agg cat ccg gaaaag gcg gtt aca ttt gtt 1314 Asp Gly Thr Val Val Ser Arg His Pro Glu LysAla Val Thr Phe Val 310 315 320 gaa aat cat gac aca cag ccg gga cag tcattg gaa tcg aca gtc caa 1362 Glu Asn His Asp Thr Gln Pro Gly Gln Ser LeuGlu Ser Thr Val Gln 325 330 335 340 act tgg ttt aaa ccg ctt gca tac gccttt att ttg aca aga gaa tcc 1410 Thr Trp Phe Lys Pro Leu Ala Tyr Ala PheIle Leu Thr Arg Glu Ser 345 350 355 ggt tat cct cag gtg ttc tat ggg gatatg tac ggg aca aaa ggg aca 1458 Gly Tyr Pro Gln Val Phe Tyr Gly Asp MetTyr Gly Thr Lys Gly Thr 360 365 370 tcg cca aag gaa att ccc tca ctg aaagat aat ata gag ccg att tta 1506 Ser Pro Lys Glu Ile Pro Ser Leu Lys AspAsn Ile Glu Pro Ile Leu 375 380 385 aaa gcg cgt aag gag tac gca tac gggccc cag cac gat tat att gac 1554 Lys Ala Arg Lys Glu Tyr Ala Tyr Gly ProGln His Asp Tyr Ile Asp 390 395 400 cac ccg gat gtg atc gga tgg acg agggaa ggt gac agc tcc gcc gcc 1602 His Pro Asp Val Ile Gly Trp Thr Arg GluGly Asp Ser Ser Ala Ala 405 410 415 420 aaa tca ggt ttg gcc gct tta atcacg gac gga ccc ggc gga tca aag 1650 Lys Ser Gly Leu Ala Ala Leu Ile ThrAsp Gly Pro Gly Gly Ser Lys 425 430 435 cgg atg tat gcc ggc ctg aaa aatgcc ggc gag aca tgg tat gac ata 1698 Arg Met Tyr Ala Gly Leu Lys Asn AlaGly Glu Thr Trp Tyr Asp Ile 440 445 450 acg ggc aac cgt tca gat act gtaaaa atc gga tct gac ggc tgg gga 1746 Thr Gly Asn Arg Ser Asp Thr Val LysIle Gly Ser Asp Gly Trp Gly 455 460 465 gag ttt cat gta aac gat ggg tccgtc tcc att tat gtt cag aaa taa 1794 Glu Phe His Val Asn Asp Gly Ser ValSer Ile Tyr Val Gln Lys 470 475 480 ggtaataaaa aaacacctcc aagctgagtgcgggtatcag cttggaggtg cgtttatttt 1854 ttcagccgta tgacaaggtc ggcatcaggtgtgacaaata cggtatgctg gctgtcatag 1914 gtgacaaatc cgggttttgc gccgtttggctttttcacat gtctgatttt tgtataatca 1974 acaggcacgg agccggaatc tttcgccttggaaaaataag cggcgatcgt agctgcttcc 2034 aatatggatt gttcatcggg atcgctgcttttaatcacaa cgtgggatcc 2084 10 483 PRT Bacillus amyloliquefaciens 10 ValAsn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65 70 7580 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 9095 Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100105 110 Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser115 120 125 Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro GlyArg 130 135 140 Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His PheAsp Gly 145 150 155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg IlePhe Lys Phe Arg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val SerSer Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val AspTyr Asp His Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly IleTrp Tyr Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp AlaAla Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val GlnAla Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala GluTyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn LysThr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe AsnLeu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg ArgLeu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp AsnIle 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly ProGln His 385 390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp ThrArg Glu Gly Asp 405 410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala LeuIle Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly LeuLys Asn Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg SerAsp Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His ValAsn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Lys 11 1458 DNABacillus sp. CDS (1)..(1458) AA560 11 cac cat aat ggt acg aac ggc acaatg atg cag tac ttt gaa tgg tat 48 His His Asn Gly Thr Asn Gly Thr MetMet Gln Tyr Phe Glu Trp Tyr 1 5 10 15 cta cca aat gac gga aac cat tggaat aga tta agg tct gat gca agt 96 Leu Pro Asn Asp Gly Asn His Trp AsnArg Leu Arg Ser Asp Ala Ser 20 25 30 aac cta aaa gat aaa ggg atc tca gcggtt tgg att cct cct gca tgg 144 Asn Leu Lys Asp Lys Gly Ile Ser Ala ValTrp Ile Pro Pro Ala Trp 35 40 45 aag ggt gcc tct caa aat gat gtg ggg tatggt gct tat gat ctg tat 192 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr GlyAla Tyr Asp Leu Tyr 50 55 60 gat tta gga gaa ttc aat caa aaa gga acc attcgt aca aaa tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile ArgThr Lys Tyr Gly 65 70 75 80 acg cgc aat cag tta caa gct gca gtt aac gccttg aaa agt aat gga 288 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala LeuLys Ser Asn Gly 85 90 95 att caa gtg tat ggc gat gtt gta atg aat cat aaaggg gga gca gac 336 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys GlyGly Ala Asp 100 105 110 gct acc gaa atg gtt agg gca gtt gaa gta aac ccgaat aat aga aat 384 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro AsnAsn Arg Asn 115 120 125 caa gaa gtg tcc ggt gaa tat aca att gag gct tggaca aag ttt gac 432 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp ThrLys Phe Asp 130 135 140 ttt cca gga cga ggt aat act cat tca aac ttc aaatgg aga tgg tat 480 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys TrpArg Trp Tyr 145 150 155 160 cac ttt gat gga gta gat tgg gat cag tca cgtaag ctg aac aat cga 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg LysLeu Asn Asn Arg 165 170 175 att tat aaa ttt aga ggt gat gga aaa ggg tgggat tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp AspTrp Glu Val Asp 180 185 190 aca gaa aac ggt aac tat gat tac cta atg tatgca gat att gac atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr AlaAsp Ile Asp Met 195 200 205 gat cac cca gag gta gtg aat gag cta aga aattgg ggt gtt tgg tat 672 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn TrpGly Val Trp Tyr 210 215 220 acg aat aca tta ggc ctt gat ggt ttt aga atagat gca gta aaa cat 720 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile AspAla Val Lys His 225 230 235 240 ata aaa tac agc ttt act cgt gat tgg attaat cat gtt aga agt gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile AsnHis Val Arg Ser Ala 245 250 255 act ggc aaa aat atg ttt gcg gtt gcg gaattt tgg aaa aat gat tta 816 Thr Gly Lys Asn Met Phe Ala Val Ala Glu PheTrp Lys Asn Asp Leu 260 265 270 ggt gct att gaa aac tat tta aac aaa acaaac tgg aac cat tca gtc 864 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr AsnTrp Asn His Ser Val 275 280 285 ttt gat gtt ccg ctg cac tat aac ctc tataat gct tca aaa agc gga 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr AsnAla Ser Lys Ser Gly 290 295 300 ggg aat tat gat atg agg caa ata ttt aatggt aca gtc gtg caa aga 960 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn GlyThr Val Val Gln Arg 305 310 315 320 cat cca atg cat gct gtt aca ttt gttgat aat cat gat tcg caa cct 1008 His Pro Met His Ala Val Thr Phe Val AspAsn His Asp Ser Gln Pro 325 330 335 gaa gaa gct tta gag tct ttt gtt gaagaa tgg ttc aaa cca tta gcg 1056 Glu Glu Ala Leu Glu Ser Phe Val Glu GluTrp Phe Lys Pro Leu Ala 340 345 350 tat gct ttg aca tta aca cgt gaa caaggc tac cct tct gta ttt tat 1104 Tyr Ala Leu Thr Leu Thr Arg Glu Gln GlyTyr Pro Ser Val Phe Tyr 355 360 365 gga gat tat tat ggc att cca acg catggt gta cca gcg atg aaa tcg 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His GlyVal Pro Ala Met Lys Ser 370 375 380 aaa att gac ccg att cta gaa gcg cgtcaa aag tat gca tat gga aga 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg GlnLys Tyr Ala Tyr Gly Arg 385 390 395 400 caa aat gac tac tta gac cat cataat atc atc ggt tgg aca cgt gaa 1248 Gln Asn Asp Tyr Leu Asp His His AsnIle Ile Gly Trp Thr Arg Glu 405 410 415 ggg aat aca gca cac ccc aac tccggt tta gct act atc atg tcc gat 1296 Gly Asn Thr Ala His Pro Asn Ser GlyLeu Ala Thr Ile Met Ser Asp 420 425 430 ggg gca gga gga aat aag tgg atgttt gtt ggg cgt aat aaa gct ggt 1344 Gly Ala Gly Gly Asn Lys Trp Met PheVal Gly Arg Asn Lys Ala Gly 435 440 445 caa gtt tgg acc gat atc act ggaaat cgt gca ggt act gtt acg att 1392 Gln Val Trp Thr Asp Ile Thr Gly AsnArg Ala Gly Thr Val Thr Ile 450 455 460 aat gct gat gga tgg ggt aat ttttct gta aat gga gga tca gtt tct 1440 Asn Ala Asp Gly Trp Gly Asn Phe SerVal Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg gta aac aaa taa 1458Ile Trp Val Asn Lys 485 12 485 PRT Bacillus sp. 12 His His Asn Gly ThrAsn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn AspGly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys AspLys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala SerGln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly GluPhe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg AsnGln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln ValTyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala ThrGlu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 GlnGlu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu ValAsp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp IleAsp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp GlyVal Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile AspAla Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp IleAsn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val AlaGlu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu AsnLys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His TyrAsn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met ArgGln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Met HisAla Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu AlaLeu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr AlaLeu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 GlyAsp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met SerAsp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn LysAla Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly ThrVal Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn GlyGly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 13 485 PRTBacillus 707 13 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe GluTrp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn SerAsp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile ProPro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala TyrAsp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg ThrLys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser LeuLys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His LysGly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val AsnPro Asn Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile GluAla Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His SerSer Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp TrpAsp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg GlyHis Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly AsnTyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro GluVal Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn ThrLeu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 IleLys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265270 Gly Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val 275280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly290 295 300 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val GlnArg 305 310 315 320 His Pro Ser His Ala Val Thr Phe Val Asp Asn His AspSer Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp PheLys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly TyrPro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His GlyVal Pro Ala Met Arg Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala ArgGln Lys Tyr Ala Tyr Gly Lys 385 390 395 400 Gln Asn Asp Tyr Leu Asp HisHis Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His ProAsn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly SerLys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp SerAsp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala AspGly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 IleTrp Val Asn Lys 485

1. A variant of a parent Termamyl-like alpha-amylase, comprising analteration at one or more positions selected from the group of: 5, 6,36, 37, 38, 39, 42, 45, 47, 63, 66, 69, 70, 71, 72, 74, 75, 76, 79, 82,83, 86, 87, 89, 93, 112, 113, 117, 120, 137, 213, 216, 220, 223, 225,226, 227, 229, 243, 245, 279, 282, 311, 321, 324, 352, 353, 354, 357,361, 362, 364, 368, 390, 395, 397, 399, 400, 401, 425, 451, 452, 453,466, 468, 470, 471, 478, wherein (a) the alteration(s) are independently(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position, (ii) a deletion of the amino acid which occupiesthe position, or (iii) a substitution of the amino acid which occupiesthe position with a different amino acid, (b) the variant hasalpha-amylase activity and (c) each position corresponds to a positionof the amino acid sequence of the parent termamyl-like alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 8 (bacilluslicheniformis alph-amylase).
 2. A variant of a parent Termamyl-likealpha-amylase, comprising one or more of the following substitutions.X1A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;X2R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;X3A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y;X4A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X13R,N,D,C,Q,E,G,H,K,M,P,S,T,W; X14A,R,D,C,G,K,M,P,W;X16R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;X17A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X18A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X20A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X23A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;X24A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X26A,D,C,E,G,H,I,L,M,F,P,S,T,W,V;X34A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;X35A,R,N,D,C,Q,E,G,H,K,M,F,P,S,T,W,Y,V; X49A,C,G,H,P,T;X50A,R,N,C,Q,E,G,H,K,M,F,P,S,W; X51A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X52A,R,D,C,Q,E,G,H,K,P; X53A,D,C,G,H,K,M,P;X61A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y; X62A,R,D,C,G,K,M,P,Y;X67A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;X68A,R,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X73A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;X84A,R,N,D,C,G,H,I,L,K,M,F,P,S,T,W,Y,V;X85A,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X88A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X91A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X92A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X96A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X106A,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;X108R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X114A,N,C,Q,E,G,H,K,F,P,S,T,W,Y; X116A,R,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;X119A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X121A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X122R,N,Q,G,H,I,L,M,F,S,T,W,Y,V; X123N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X124N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X125R,N,Q,E,G,I,K,M,F,S,T,W,Y;X126N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X127A,R,N,D,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X128A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,W,Y,V;X129A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;X130A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;X131A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;X132R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y; X133R,N,D,C,M,T,W,V;X134A,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X136A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X138R,N,D,Q,E,G,I,K,M,P,S,T,W,V;X145A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,Y,V;X147A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;X148A,R,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;X149A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V; X150A,D,C,G,M,P,W,Y;X152A,R,N,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;X153A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X154A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X155A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X156A,C,Q,E,G,I,L,M,F,P,S,T,W,V; X157R,I,L,M,F,P,S,T,W,Y,V;X158R,M,P,W,Y; X164R,I,L,M,F,P,S,T,W,Y,V;X165A,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;X167A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;X168A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,V;X169A,R,N,D,C,Q,E,G,H,M,P,S,W,Y,V;X170A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X171A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;X172A,N,D,C,Q,E,G,I,L,M,F,P,T,W,Y,V; X173A,N,D,C,Q,E,G,H,M,P,S,W,Y,V;X176A,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X179D,C,Q,E,H,I,L,K,M,F,P,W,Y,V; X180A,G,I,L,M,F,P,W,Y,V; X181G;X182A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X184I,L,M,F,P,W,Y,V;X185R,I,L,M,F,P,S,T,W,Y,V; X188A,R,N,Q,G,H,L,M,F,W,V;X189A,R,N,G,H,I,L,M,F,P,S,T,W,Y,V; X190N;X191A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X193A,R,G,M,P,W,Y;X196A,N,Q,G,H,I,L,M,P,S,T,W,V; X198A,R,G,M,P,W; X204R,L,M,F,P,T,W,Y,V;X205A,G,H,I,L,M,F,P,W,Y,V; X206R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X209R,P,S,W,Y; X210A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V; X211E;X214A,D,C,Q,E,G,I,L,M,F,P,S,T,Y,V;X217A,R,N,D,C,Q,G,H,I,L,M,F,P,S,T,W,Y;X218A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X221A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X222A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,W,Y,V;X234A,D,C,G,H,I,K,M,F,P,S,T,W,Y,V;X235A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X237A,G,H,I,L,M,F,W,Y,V;X239G,H,I,L,M,F,P,S,T,Y,V; X242G,I,L,M,F,S,T,W,Y,V;X246A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X247R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;X249A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X250A,R,N,D,C,E,H,I,L,K,M,P,T,W,Y,V;X251R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X252A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;X253R,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y;X254A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X255A,R,D,C,G,H,I,L,K,M,F,S,T,W,Y,V;X257A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X261A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X263A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V; X265C,Q,E,H,I,L,M,F,P,W;X266A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X267A,R,N,D,C,Q,E,G,H,K,P,S,T,W,Y,V;X268A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X269A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X271A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X272A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X275A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;X276A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X278A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X280A,R,D,C,E,G,H,I,L,K,M,F,P,W,Y,V; X290W,Y;X291A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X293A,R,N,G,I,L,M,P,S,T,W,V;X294R,N,Q,G,H,I,L,M,F,P,S,T,W,Y; X297A,G,H,I,L, M, F,P,W,Y,V;X298G,H,I,L,M,F,P,S,T,W,Y,V; X299A,G,H,M,P,S,T;X300A,C,G,H,I,L,M,F,P,T,W,Y,V; X301 N,Q,H,I,L,M,F,P,S,T,W,Y,V;X302R,M,P,W,Y; X303R,I,L,M,F,P,S,T,W,Y,V; X305G,I,L,M,F,P,S,T,W,Y,V;X306Q,G,H,I,L,M,F,P,S,T,W,Y,V;X308A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X309N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X310A,R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V; X314A,D,C,E,G,I,L,M,F,P,W,Y,V;X315A,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X316A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;X317R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X318A,R,N,D,C,Q,E,G,H,I,K,P,S,W,Y,V;X319A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X328A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X332A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X333A,N,D,C,G,I,M,F,P,S,T,Y,V; X334R,N,C,Q,E,G,H,K,M,F,P,S,W,Y;X335R,D,C,Q,E,H,I,L,K,M,F,P,W,Y,V;X336A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X337A,R,N,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X338A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; X340I,L,M,F,P,S,T,W,Y,V;X341A,R,D,C,G,H,I,L,K,M,F,W,Y,V; X342R,I,L,M,F,P,S,T,W,Y,V;X345R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;X355A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; X358A,R,D,C,G,K,M,P,W,Y;X363A,R,D,C,G,K,M,P,W,Y; X370A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X371A,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X373A,R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;X374A,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;X375A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,V;X376A,RN,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X378R,N,D,C,Q,E,G,H,I,L,K,M,F,S,T,W,Y,V;X379A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;X381A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X389A,D,C,G,H,M,F,P,S,T,W,Y,V; X393A,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X394A,R,D,C,G,K,M,P,W,Y; X396A,R,D,C,G,K,M,P,W,Y;X398A,R,N,D,C,Q,E,G,H,I,L,M,F,W,Y,V; X402R,C,G,K,M,P,W,Y;X403A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X404R,I,L,M,F,P,S,T,W,Y,V;X405R,G,H,I,L,M,F,P,W,Y,V; X406A,R,G,H,I,M,F,P,Y,V;X407A,R,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X408A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;X413A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X414A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X415A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X416A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X417A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V;X418A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X419A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X420A,N,D,C,E,G,H,I,L,K,M,F,S,T,W,Y,V;X421A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;X422A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X431A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X432G,H,I,L,M,F,P,S,T,W,Y,V;X433A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X434A,R,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X435Q,G,H,I,L,M,F,P,T,W,Y,V;X436A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X437A,N,D,C,Q,E,G,H,i,L,K,M,F,P,S,T,W,Y,V; X439A,R,D,C,G,K,M,P,W,Y;X442A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;X443A,R,N,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;X444A,C,G,H,I,L,M,F,P,S,T,W,Y,V;X445A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X446A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X447A,N,D,C,G,H,I,L,M,F,P,S,T,W,Y,V;X448A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X450A,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,V;X454A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X455A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;X456A,D,C,E,G,H,I,L,M,F,P,S,T,W,Y,V;X457A,R,N,D,C,Q,E,G,H,I,L,K,M,F,W,Y,V;X458A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X459R,N,D,C,Q,E,G,H,I,L,K,M,F,S,W,Y,V;X460A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X461A,R,N,D,C,Q,E,G,H,I,L,M,F,P,S,W,Y,V;X463A,R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;X464A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X465A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X467A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X469A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;X473N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X474A,R,G,H,I,L,M,F,P,W,Y,V;X475A,N,Q,G,H,I,L,M,F,P,S,T,W,Y,V; X476G,H,I,L,M,F,P,S,T,W,Y,V;X482A,N,D,C,G,H,I,L,M,F,S,T,W,Y,V;X483A,N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V wherein (a) the variant hasalpha-amylase activity and (b) each position corresponds to a positionof the amino acid sequence of the parent Termamyl-like alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 8 (Bacilluslicheniformis alph-amylase).
 3. A variant of a parent Termamyl-likealpha-amylase, comprising one or more of the following substitutions:X7A,R,N,D,C,Q,E,G,H,K,M,P,S,Y,V; X8C,M X9A,R,N,D,C,Q,G,H,M,P,S,T,W,Y,V;X11A,N,D,C,Q,G,H,I,L,M,P,S,T,W,Y,V;X12A,R,N,D,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X19A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X21A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X22A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X25A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X32A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X40A,R,N,D,C,Q,E,G,H,l,L,K,M,F,P,S,T,W,Y,V;X41A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X46A,R,D,C,G,K,M,P,W,Y;X48R,N,D,C,Q,E,G,H,K,M,F,P,W,Y;X55A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X57R,N,D,C,Q,E,G,H,K,M,P,W;X58A,R,N,D,C,Q,E,G,H,K,M,S,T,W,Y;X60A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X77A,R,D,C,G,K,M,P,W,Y;X95A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X97A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X98A,R,D,C,G,K,M,P,W,Y;X99R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X100A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;X101A,N,C,Q,G,I,L,M,P,S,T,W,Y,V; X102N,D,C,Q,E,H,I,L,M,F,P,W,Y,V;X103A,N,D,C,Q,E,G,M,P,S,W,Y; X105A,N,C,Q,G,H,I,L,M,P,S,T,Y,V;X107R,N,D,Q,E,H,K,M,F,P,W,Y; X115R,N,D,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;X118R,N,C,Q,E,H,I,L,K,M,F,P,S,T,W,Y,V;X135A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X139A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X141A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X143A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;X151A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V;X159A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,V;X160A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X161A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X162A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X163A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X166A,R,N,D,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V; X175A,R,D,C,G,K,M,P,W,Y;X177A,N,D,C,Q,E,H,I,L,K,M,P,S,T,W,Y,V;X183A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X186A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X187A,R,C,Q,E,G,H,I,L,K,M,F,P,W,Y,V;X192A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X199R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X200A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X202A,R,D,C,Q,E,.G,H,I,L,K,M,F,P,S,T,W,Y,V; X203A,R,D,C,G,K,M,P;X208A,R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V; X212A,N,C,Q,G,H,M,P,S,T,V;X215A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X219A,R,D,C,G,K,M,P,W,Y;X228A,R,N,D,C,Q,E,G,H,I,L,K,M,P,S,T,W,Y,V;X230A,R,N,D,C,Q,E,G,L,M,P,S,T,W,Y,V; X233R,N,C,Q,E,G,H,I,K,M,P,S,T,W,Y;X236A,C,Q,G,H,I,M,P,S,T,V; X238A,R,N,D,C,Q,E,G,H,I,K,M,P,S,T,W,Y,V;X240A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X241A,N,C,Q,G,H,P,S,T,V;X244A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X248A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X256C,M;X258A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X259A,R,N,D,C,Q,E,G,H,K,M,P,S,T,W,Y,V;X260R,N,D,C,Q,E,H,I,L,K,M,F,P,T,W,Y,V; X262A,R,D,C,G,K,M,P,W,Y;X270A,N,C,Q,G,I,L,M,F,P,S,T,W,Y,V; X273A,R,D,C,G,K,M,P,Y;X274A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X277A,R,N,D,C,Q,E,G,H,K,M,P,S,W,Y,V;X281A,R,N,D,C,Q,E,G,K,M,P,S,T,W,Y,V;X283A,R,N,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;X284A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,Y,V;X285A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;X286A,R,D,C,Q,E,G,H,I,K,M,F,P,S,T,W,Y,V;preferablyX286N,C,Q,I,L,M,P,T,V,Y,F; X287R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X288A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X289A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X292A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X295A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X296A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X304C,M;X307A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X312A,N,C,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X313A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X320R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X322R,N,D,C,Q,E,G,H,L,K,M,F,P,S,T,W,Y,V;X323A,R,N,D,C,Q,E,G,I,L,K,M,F,P,S,T,W,Y,V;X325A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X326A,R,N,C,Q,E,G,M,P,S,T,W;X327A,R,C,G,H,I,L,K,M,P,S,T,W,Y,V;X329A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,T,W,Y,V;X331N,D,C,Q,E,G,H,I,L,M,F,P,S,T,W,Y,V;X339A,R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X343A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,Y,V;X344A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X346A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X347A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X349R,N,D,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X350A,R,N,C,Q,G,H,I,L,K,M,F,P,S,T,Y,V;X359R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X360N,D,Q,G,H,I,L,M,F,P,S,T,W,Y,V;X369A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X377A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X380A,R,N,D,C,Q,E,G,H,K,P,S,W,Y,V;X387A,R,N,D,C,Q,E,G,H,L,K,M,P,S,T,W,Y,V; X409N,C,Q,E,G,H,M,P,S,T,W,Y,V;X410A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X411A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X412R,N,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X423A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X424A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X426A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X427A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V; X428A,N,D,Q,E,G,H,I,M,F,P,S,W,Y,V;X429A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X430A,R,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V; X438C,M;X440R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X441A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X449A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X462A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X472A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X477A,R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;X479A,R,N,D,Q,E,G,H,I,L,K,M,F,P,S,W,Y,V; X480A,R,D,C,G,K,M,P,W,Y;X481A,R,N,D,C,Q,E,G,H,K,M,P,S,T,Y,V wherein (a) the variant hasalpha-amylase activity and (b) each position corresponds to a positionof the amino acid sequence of the parent Termamyl-like alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 8 (Bacilluslicheniformis alpha-amylase).
 4. A variant of a parent Termamyl-likealpha-amylase, comprising an alteration at one or more positionsselected from the group of: A1 insertion; L3 insertion; N4 insertion;N17 insertion; D18 insertion; Q20 insertion; R23 insertion; R24insertion; D28 insertion; Y56 insertion; L61 insertion or deletion; Y62insertion; F67 insertion or deletion; H68 insertion; K80 insertion ordeletion; G81 insertion or deletion; Q84 insertion; S85 insertion; H91insertion or deletion; S92 insertion or deletion; K106 insertion ordeletion; D110 insertion or deletion; D114 deletion; E119 insertion ordeletion; D121 insertion; P122 insertion; A123 insertion; D124insertion; R125 insertion; N126 insertion; R127 insertion; I129insertion; G131 insertion; L134 insertion; K136 insertion; N172insertion; E185 insertion; L196 insertion or deletion; P206 insertion ordeletion; T217 insertion; W218 insertion; D231 insertion or deletion;A232 insertion or deletion; H235 insertion or deletion; N246 insertion;H247 insertion; R249 insertion; K251 insertion; F257 insertion ordeletion; N278 insertion; G310 insertion or deletion; H316 insertion;P317 insertion; D328 insertion or deletion; G332 insertion or deletion;E355 insertion or deletion; Y358 insertion; Y363 insertion; Y367insertion; K370 insertion; S373 insertion; R375 insertion; E376insertion; K381 insertion; H382 insertion; R391 insertion or deletion;Y396 insertion; R413 insertion or deletion; E414 insertion or deletion;G415 insertion or deletion; D416 insertion; S417 insertion; S418insertion; V419 insertion; A420 insertion; N421 insertion; S422insertion or deletion; Y439 insertion; A445 insertion or deletion; G446insertion or deletion; T448 insertion or deletion; H450 insertion; G454insertion or deletion; N455 insertion; E458 insertion; P459 insertion;V460 insertion; V461 insertion; N463 insertion; S464 insertion; E465insertion; W467 insertion; wherein (a) the alteration(s) areindependently (as specified above): (i) an insertion of an amino aciddownstream of the amino acid which occupies the position, or (ii) adeletion of the amino acid which occupies the position, (b) the varianthas alpha-amylase activity and (c) each position corresponds to aposition of the amino acid sequence of the parent Termamyl-likealpha-amylase having the amino acid sequence shown in SEQ ID NO: 8(Bacillus licheniformis alph-amylase).
 5. A variant of a parentTermamyl-like alpha-amylase, comprising an alteration at one or morepositions selected from the group of: L7 insertion or deletion; M8insertion; Y10 insertion; F11 insertion; E12 insertion or deletion; M15insertion; G19 insertion; H21 insertion; W22 insertion; L25 insertion;V40 insertion or deletion; W41 insertion; P43 insertion or deletion; P44insertion or deletion; Y46 insertion; G55 insertion; Y59 insertion; Y77insertion; G78 insertion or deletion; L90 insertion or deletion; I95insertion; V97 insertion; Y98 insertion; G99 insertion; D100 insertion;V101 insertion; V102 insertion; H105 insertion or deletion; A109insertion or deletion; V115 insertion or deletion; V118 insertion ordeletion; I135 insertion; T139 insertion or deletion; F141 insertion ordeletion; Y195 insertion; V208 insertion or deletion; W215 insertion;Y219 insertion; I236 insertion or deletion; F238 insertion or deletion;F240 insertion or deletion; W244 insertion; V248 insertion; M256insertion; T258 insertion or deletion; V259 insertion or deletion; V312insertion or deletion; V313 insertion or deletion; S320 insertion; T322insertion or deletion; F323 insertion or deletion; D325 insertion ordeletion; N326 insertion; H327 insertion or deletion; Q330 insertion ordeletion; P331 insertion or deletion; Y348 insertion; A349 insertion ordeletion; F350 insertion or deletion; P359 insertion or deletion; Q360insertion; D365 insertion or deletion; M366 insertion; T369 insertion;I377 insertion; I384 insertion or deletion; L388 insertion or deletion;G423 insertion or deletion; L424 insertion or deletion; M438 insertion;G441 insertion or deletion; W449 insertion; I462 insertion; I479insertion or deletion; Y480 insertion; V481 insertion or deletion;wherein (a) the alteration(s) are independently (as specified above):(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position, or (ii) a deletion of the amino acid whichoccupies the position, (b) the variant has alpha-amylase activity and(c) each position corresponds to a position of the amino acid sequenceof the parent Termamyl-like alpha-amylase having the amino acid sequenceshown in SEQ ID NO: 8 (Bacillus licheniformis alph-amylase).
 6. Thevariant of any of claims 1-5, wherein the parent Termamyl-likealpha-amylase is derived from a strain of B. licheniformis (SEQ ID NO:8), B. amyloliquefaciens (SEQ ID NO: 10), B. stearothermophilus (SEQ IDNO: 6), Bacillus sp. (SEQ ID NO: 12 (M560), Bacillus sp. (SEQ ID NO: 2(SP690)); Bacillus sp. (SEQ ID NO: 4 (SP722); Bacillus sp. #707alpha-amylase (SEQ ID NO: 13); KSM-AP1378.
 7. The variant of any ofclaims 1 to 6, wherein the parent Termamyl-like alpha-amylase has anamino acid sequence which has a degree of identity to SEQ ID NO: 8 of atleast 60%, preferably 70%, more preferably at least 80%, even morepreferably at least about 90%, even more preferably at least 95%, evenmore preferably at least 96%, even more preferably at least 97%, evenmore preferably at least 98%, and even more preferably at least 99%. 8.The variant of any of claims 1-7, wherein the parent Termamyl-likealpha-amylase is encoded by a nucleic acid sequence (DNA), whichhydridizes under low, preferably medium, preferred high stringencyconditions, with the nucleic acid sequence of SEQ ID NO:
 7. 9. Thevariant of claims 1-8, which variant has alpha-amylase activity andexhibits an alteration in at least one of the following propertiesrelative to said parent alpha-amylase: altered substrate specificity,substrate binding, substrate cleavage pattern, thermal stability, pHactivity profile, pH stability profile, stability towards oxidation,Ca²⁺ dependency, reduced and increased pI and improved wash performance,specific activity, stability under high temperature and/or low pHconditions, in particular at low calcium concentrations and/or inparticular at high temperatures from 70-120□C. and/or low pH in therange from pH 4-6
 10. A DNA construct comprising a DNA sequence encodingan alpha-amylase variant according to any one of claims 1-9.
 11. Arecombinant expression vector which carries a nucleic acid sequence(DNA) construct according to claim
 10. 12. A cell which is transformedwith a DNA construct according to claim 10 or a vector according toclaim
 11. 13. A cell according to claim 12, which is a microorganism,preferably a bacterium or a fungus, in particular a gram-positivebacterium, such as Bacillus subtilis, Bacillus licheniformis, Bacilluslentus, Bacillus brevis, Bacillus stearothermophilus, Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacilluscirculans, Bacillus lautus or Bacillus thuringiensis.
 14. A compositioncomprising an alpha-amylase variant of any of claims 1-9.
 15. Thecomposition of claim 14, wherein the composition further comprisesglucoamylase, pullulanase and/or phytase.
 16. A detergent compositioncomprising an alpha-amylase variant according to any of claims 1-9. 17.A detergent composition of claim 16, which additionally comprisesanother enzyme such as a protease, a lipase, a peroxidase, anotheramylolytic enzyme, glucoamylase, maltogenic amylase, CGTase, mannanase,cutinase, laccase and/or a cellulase.
 18. Use of an alpha-amylasevariant according to any of claims 1-9 or a composition according toclaim 15 for starch liquefaction, in particular for syrup or ethanolproduction.
 19. Use of an alpha-amylase variant according to any one ofclaims 1-9 or a composition according to claim 15 for washing and/ordishwashing.
 20. Use of an alpha-amylase variant of any one of claims1-9 or a composition according to claim 15 for textile desizing.